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. 2014 Oct 17;143(1):136–146. doi: 10.1093/toxsci/kfu217

Formation of a Protein Corona on Silver Nanoparticles Mediates Cellular Toxicity via Scavenger Receptors

Jonathan H Shannahan *, Ramakrishna Podila †,‡, Abdullah A Aldossari *, Hilary Emerson §, Brian A Powell §, Pu Chun Ke †, Apparao M Rao †,‡, Jared M Brown *,1
PMCID: PMC4274384  PMID: 25326241

Abstract

Addition of a protein corona (PC) or protein adsorption layer on the surface of nanomaterials following their introduction into physiological environments may modify their activity, bio-distribution, cellular uptake, clearance, and toxicity. We hypothesize that silver nanoparticles (AgNPs) will associate with proteins common to human serum and cell culture media forming a PC that will impact cell activation and cytotoxicity. Furthermore, the role of scavenger receptor BI (SR-BI) in mediating this toxicity was evaluated. Citrate-suspended 20 nm AgNPs were incubated with human serum albumin (HSA), bovine serum albumin (BSA), high-density lipoprotein (HDL), or water (control) to form a PC. AgNPs associated with each protein (HSA, BSA, and HDL) forming PCs as assessed by electron microscopy, hyperspectral analysis, ζ-potential, and hydrodynamic size. Addition of the PC decreased uptake of AgNPs by rat lung epithelial and rat aortic endothelial cells. Hyperspectral analysis demonstrated a loss of the AgNP PC following internalization. Cells demonstrated concentration-dependent cytotoxicity following exposure to AgNPs with or without PCs (0, 6.25, 12.5, 25 or 50 μg/ml). All PC-coated AgNPs were found to activate cells by inducing IL-6 mRNA expression. A small molecule SR-BI inhibitor was utilized to determine the role of SR-BI in the observed effects. Pretreatment with the SR-BI inhibitor decreased internalization of AgNPs with or without PCs, and reduced both cytotoxicity and IL-6 mRNA expression. This study characterizes the formation of a PC on AgNPs and demonstrates its influence on cytotoxicity and cell activation through a cell surface receptor.

Keywords: nanotoxicology, epithelial cells, endothelial cells, in vitro toxicity, hyperspectral microscopy, darkfield microscopy

Nanoparticles (NPs) are increasingly being utilized in a variety of fields including inclusion into commercial products and biomedical applications. Following their introduction into a biological environment the surface of NPs associate with an assortment of biomolecules including proteins, peptides, and lipids forming a NP-protein layer referred to as the protein corona (PC) (Lynch et al., 2007; Monopoli et al., 2012). The PC imparts a unique physicochemical identity to NPs by altering hydrodynamic size, shape, charge, and interfacial composition, which can modify biodistribution, activity, clearance, and toxicity (Riviere, 2009; Walkey and Chan, 2012). Based on these modifications the PC has been shown to affect the uptake and distribution of NPs (Chithrani and Chan, 2007; Lesniak et al., 2012; Lunov et al., 2011; Safi et al., 2011). Specifically, it has been found that the association of human serum albumin, IgG, or transferrin reduces the uptake of 20 and 110 nm citrate-coated AgNPs by HEK cells, while association of IgG with silica-coated AgNPs increased uptake (Monteiro-Riviere et al., 2013). Studies have also demonstrated that the addition of fibrinogen, C3, and other proteins individually to NPs and liposomes enhances their cellular uptake (Clift et al., 2010; Moghimi et al., 2001). Conversely, the PC may inhibit interactions with cell surface receptors thus reducing receptor-mediated NP uptake (Serda et al., 2009; Walkey and Chan, 2012). These findings suggest that the PC may influence biodistribution and cellular uptake of NP modifying cellular and physiological responses. Thus, the PC may alter the therapeutic efficacy of numerous NPs and contribute to unintended toxicity.

Silver nanoparticles (AgNPs) have been incorporated as antimicrobial agents in medical devices, water treatment, textile engineering, and have been integrated into the surfaces of many household appliances, and food storage containers (Lin et al., 2012; Martinez-Gutierrez et al., 2013). AgNPs can be manufactured in different sizes and typically are suspended in either citrate or polyvinylpyrrolidone, which increases the steric separation and dispersion of particles in solution, allowing for a more stable suspension. Recently, we have demonstrated that these suspension materials result in the formation of unique PCs in cell culture media. However, it is unknown how the PC may influence toxicity. AgNPs have been reported to induce cytotoxicity, oxidative stress, and apoptosis in different cell types, including macrophages, monocytes, and epithelial cells (Foldbjerg et al., 2011; Lanone et al., 2009; Park et al., 2007). Furthermore, AgNPs have also been found to impair membrane function of rat endothelial cells (Grosse et al., 2013). The formation of the NP-PC is dependent on the physicochemical properties of the NP as well as the physiological environment. Based on its high abundance within serum, human serum albumin (HSA) has been demonstrated to associate with many NPs (Alkilany et al., 2009; Monteiro-Riviere et al., 2013; Podila et al., 2012). Furthermore, bovine serum albumin (BSA) is a major component of serum utilized in cell culture for the toxicity assessment of NPs and also has been shown to associate with many NPs (Shannahan et al., 2013a, b). Studies have shown that albumin can bind to different NPs such as Au and Ag (Alkilany et al., 2009; Monteiro-Riviere et al., 2013). High-density lipoprotein (HDL) has been reported to be associated with NPs such as quantum dots (Prapainop and Wentworth, 2011) and has been shown to preferentially associate with AgNPs (Shannahan et al., 2013b). Based on the physiological abundance of these proteins and their likely interactions with NPs, in this study we assessed differences in AgNP-induced toxicity due to the addition of HSA, BSA, and HDL.

Scavenger receptors have been shown to mediate the uptake of AgNPs by macrophages and subsequent apoptosis (Singh and Ramarao, 2012). Scavenger receptor BI (SR-BI) is a multi-ligand receptor that binds modified LDL and HDL mediating the uptake of lipids and cholesterol by cells and is also known to bind serum albumin (Acton et al., 1994; Baranova et al., 2005; Krieger and Herz, 1994; Landschulz et al., 1996; Rigotti et al., 1997a, b). SR-BI has also been reported to play a role in the recognition of pathogens, hepatitis C virus, mycobacteria, and other foreign materials (Philips et al., 2005; Scarselli et al., 2002; Vishnyakova et al., 2003, 2006). SR-BI is expressed in different types of tissues and cells such as adrenal gland, macrophages, platelets, epithelial, and endothelial cells (Acton et al., 1996; Duncan et al., 2002; Krieger, 1999; Rigotti et al., 1997a; Valacchi et al., 2011). SR-BI is the primary scavenger receptor expressed on the surface of endothelial and epithelial cells (Dieudonne et al., 2012; Kzhyshkowska et al., 2012; Uittenbogaard et al., 2000). It is unclear how the addition of the PC may affect the uptake of AgNPs through the SR-BI receptor.

We hypothesize that AgNPs will associate with common proteins found in human serum and cell culture media to form PCs, which will influence cell activation and cytotoxicity in vitro. Furthermore, SR-BI is known to facilitate the uptake of HDL and albumin by cells and scavenger receptors have been shown to be responsible for the uptake of AgNPs and subsequent toxicity (Acton et al., 1994; Baranova et al., 2005; Krieger and Herz, 1994; Landschulz et al., 1996; Rigotti et al., 1997a, b; Singh and Ramarao, 2012). Therefore, we hypothesize that the addition of PCs consisting of albumin and HDL will modify AgNP cellular uptake and toxicity via SR-BI. To address this hypothesis, the ability of HSA, BSA, and HDL to associate with citrate-suspended AgNPs to form a PC was examined. To determine differences in cellular responses and toxicity based on the additions of these simplified PCs, rat lung epithelial (RLE) and rat aortic endothelial (RAEC) cells were exposed. Finally, to specifically evaluate the role of SR-BI in the toxicity induced by AgNPs with or without PCs, we utilized a selective small molecule SR-BI inhibitor.

MATERIALS AND METHODS

Protein corona formation on AgNPs

Citrate-suspended AgNPs with a diameter of 20 nm were procured from NanoComposix by the National Centers for Nanotechnology Health Implications Research (NCNHIR) in suspensions of citrate (C6H5O73−) at a concentration of 1 mg/ml. AgNPs were incubated at 10°C for 8 h on a rotator in the presence of either HSA or BSA at a final concentration of 357 μg/ml, HDL at a final concentration of 893 μg/ml, or deionized (DI) water (control). After incubation AgNPs were centrifuged at 14 000 rpm (20 817 g) for 10 min at 4°C and resuspended to 1 mg/ml with DI water. AgNPs were incubated with proteins for 8 h to ensure a stable equilibrium was reached and at 10°C to prevent any entropy-driven or thermally induced unfolding of proteins. The concentrations of proteins were selected to be in excess and to induce protein-protein interactions.

AgNP and protein corona characterization

The hydrodynamic size and ζ-potentials of all PC-coated AgNPs and control uncoated AgNPs were characterized in DI water with AgNPs at a concentration of 50 μg/ml using a ZetaSizer Nano (Malvern, Malvern UK). Characteristics of AgNPs were evaluated at 50 μg/ml to represent the highest dose utilized within our studies (n = 3 per particle type). The formation of the PC was further confirmed and characterized via transmission electron microscopy (TEM, Hitachi H7600) after the AgNPs were incubated with HSA, BSA, and HDL as described above, air-dried on copper grids, followed by negative staining with phosphotungstic acid for 45 min prior to imaging. Hyperspectral enhanced dark field microscopy (Cytoviva, Auburn AL) was utilized to characterize shifts in spectra following association of the PC. Following formation of the individual PCs, particles were loaded onto premium clean microscope slides and mean spectrums were created utilizing pixels with an intensity >1000. Mean spectrums were then compared with AgNPs without a PC to determine changes due to the association of individual PCs. Addition of the PC was confirmed by red shifts in spectrums of AgNPs indicative of protein coating of the nanoparticle surface (Podila et al., 2012). Differences in AgNP dissolution were evaluated by incubating AgNPs with proteins (HSA and BSA 357 μg/ml and HDL 893 μg/ml, respectively) for 8 h in water at 10°C and then centrifuging for 10 min at 14 000 rpm (20 817 g) and collecting the supernatant for inductively coupled plasma-mass spectrometry (ICP-MS) analysis of Ag+ content.

Alterations in protein structure due to protein corona formation

AgNPs were incubated as described before with HSA, BSA, HDL, or DI water (control); however, AgNPs were not centrifuged to remove unassociated proteins. This was done to provide a sufficient quantity of proteins for instrument detection and to allow for comparisons with pure proteins. Changes in protein structure upon association with AgNPs were evaluated in each sample and compared with pure protein samples by circular dichroism spectroscopy (Jasco J-810 spectropolarimeter) at room temperature over a wavelength range of 200–300 nm in quartz cuvettes. A total of 6 scans were averaged at a speed of 50 nm/min and the background signals form AgNPs alone were removed accordingly. The ellipticity values (θ, in mdeg) provided by the instrument were converted to standard unit of deg·cm2/dmol ([θ]) using the equation [θ]=(θ×M0)/(10000×Csoln×L), where M0 is the mean residue molecular weight (118 g/mol), Csoln is the protein concentration in solution (in g/ml), and L is the path length through the buffer (1 cm). The percents of protein secondary structures were rendered by both CONTINLL and CDSStr algorithms.

Cell culture

RLE (ATCC, Manassas, VA) were cultured in Hams F12 media containing L-glutamine (2 mM), bovine pituitary extract (0.01 mg/ml), insulin (0.005 mg/ml), insulin-like growth factor (2.5 ng/ml), transferrin (1.25 µg/ml), EGF-murine (2.5 ng/ml) and 10% FBS. RAEC were cultured in rat endothelial cell growth media (Cell Applications Inc., San Diego, CA). Cell lines were maintained in flasks under standard conditions at 37°C and 5% CO2. For the assessment of the PC and its role in cellular responses, all experiments were performed utilizing serum-free media. The removal of serum from media allows for the evaluation of the intentionally formed PC without the addition of a secondary PC within the cell culture system. The use of serum-free media limits study of the PC and cellular responses to acute time points.

Cellular uptake of AgNPs

RLE and RAEC cells were grown to 90% confluency in 24 well plates (Costar) and were pretreated for 30 min with either serum-free media (control) or an SR-BI inhibitor 2 -(2-butoxyethyl)-1-cyclopentanone thosemicarbazone (Blt2) (Chembridge Corp., San Diego, CA) at 50 μM in serum-free media. Cells were then washed with phosphate buffered saline (PBS), and then exposed to AgNPs (0 or 25 μg/ml) with or without PCs for 2 h. The concentration and time point selected for the evaluation of cellular uptake was chosen based on previous research examining the uptake of AgNPs by human epidermal keratinocytes (Monteiro-Riviere et al., 2013). Following exposure, cells were washed with PBS and detached with 250 μl of trypsin. Trypsin was neutralized with an equal volume of cell-specific media and cells were collected. Changes in uptake were assessed by alterations in side scatter shift through flow cytometry (Accuri C6 Flow Cytometer, BD Biosciences, San Jose, CA). To compare between studies, AgNP-induced changes in side scatter shift were normalized to serum-free media-treated controls and expressed as fold change. Qualitative assessment of uptake was performed by growing cells on microscope chamber slides and following the same exposure protocol. After being fixed with 2% paraformaldehyde cells were evaluated by enhanced dark field microscopy. AgNPs within cells were assessed by focusing on the nucleus of cells, which was stained with . Finally, cells were again exposed following the same protocol, washed 3 times with PBS and differences in uptake were determined by ICP-MS analysis of Ag content. All samples including as purchased AgNPs and the cells exposed to NPs were dissolved in 6 ml of 2% HNO3. Subsequently, the Ag concentration was determined with ICP–MS (X series II, Thermo Scientific) using an internal standard containing Li, Y, and In with a detection limit of 6 ppb (corrected for sample dilution).

Intracellular modifications to the PC

RLE and RAEC cells were grown on microscope chamber slides and were exposed to AgNPs (0 or 25 µg/ml) with or without PCs for 2 h in serum-free media and fixed with 2% paraformaldehyde. Hyperspectral dark field microscopy (Cytoviva, Auburn AL) was utilized to evaluate changes in AgNP-PC spectrums. AgNPs within cells were assessed by focusing on the 4’,6 - diamidino - 2- phenylindole (DAPI)-stained nucleus of the cell and a hyperspectral image was collected at a magnification of 100 x. To generate spectral profiles, a minimum of 1000 pixels of AgNPs were collected to form a region of interest that was used to create a mean spectra. This spectrum was then normalized and compared with the normalized original spectrum of the corresponding AgNP. These normalized spectra allowed for comparisons of peaks between AgNPs and intracellular AgNPs.

Assessment of AgNP cytotoxicity

RLE and RAEC cells were grown to 90% confluency in 96 well plates (Costar) and were exposed to increasing concentrations of AgNPs (0, 6.25, 12.5, 25, or 50 µg/ml) with or without PCs for 3 and 6 h in serum-free media. Due to the use of serum-free media, later time points were not evaluated. The concentrations of AgNPs used to evaluate cytotoxicity were selected based on previous in vitro examination of other nanoparticles (Xia et al., 2013). Changes in cell viability were assessed via the 3 - (4,5 - dimethylthiazol - 2 - yl)- 5- (3- carboxmethoxyphenyl) - 2- (4 - sulfophenyl) - 2H - tetrazolium (MTS) assay (Promega, Madison, WI) via manufacturer’s instructions using a spectrophotometer (BioTek Synergy HT, BioTek, Winooski, VT). Based on this dose-response study a concentration of 50 µg/ml was utilized for all subsequent in vitro experiments. To determine possible scavenger receptor-mediated cytotoxicity, RAEC and RLE were exposed to 50 µg/ml of AgNPs with or without PCs in 96 well plates for 3 h following pretreatment with a scavenger SR-BI inhibitor (Blt-2) at 50 µM (Chembridge Corp.). Cytotoxicity was assessed via lactate dehydrogenase activity in the supernatant; however due to limitations in this measurement for NPs, data were only used to confirm results from the MTS assay and are not shown (Promega, Madison, WI).

AgNP-induced cell activation

The ability of the AgNPs and the individual PCs to activate RLE and RAEC cells was assessed by pretreatment with or without Blt-2 (50 µM) for 30 min then exposure to AgNPs (50 µg/ml) with or without PCs for 1 h and measuring interleukin-6 (IL-6) mRNA expression. The time point of 1 h was selected to investigate surface receptor-mediated cell activation. Total RNA was isolated from cells using Direct-zol RNA MiniPrep (Zymo Research Corp., Irvine, CA) via manufacturer’s instructions and quantified via nanodrop (Nanodrop 2000c Spectrophotometer, Thermo Scientific). Total RNA (1 µg) was reverse transcribed to cDNA using an iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA). Quantitative real-time PCR was performed for the inflammatory marker IL-6 and reference gene GAPDH using SsoAdvancedTM SYBR Green Supermix (Bio-Rad) and QuantiTect primer assays (Qiagen, Balencia, CA). Relative mRNA fold changes were calculated considering serum-free media-exposed cells as control and normalized to GAPDH as the internal reference.

Statistical analysis

All data are presented as mean ± SEM and consist of 4–10 experiments. Each experimental group was run in triplicate and averaged together to provide a value for each experiment. Data were then analyzed by one-way ANOVA, with differences between groups assessed by Tukey post hoc tests. Pearson’s r correlation tests were run to compare cellular uptake to various other parameters (Supplementary Data). All graphs and analysis were performed using GraphPad Prism 5 software (GraphPad, San Diego, CA). Statistical significance was determined when P value was found to be ≤0.05 between groups.

RESULTS

AgNP-protein corona characterization

AgNPs were found to readily associate with HSA, BSA, and HDL forming PCs. Addition of all PCs (HSA, BSA, and HDL) was found to increase the hydrodynamic size of AgNPs (Table 1). The original citrate suspension of AgNPs demonstrated a negative ζ-potential, which was reduced after the addition of each PC, with HDL causing the greatest reduction in charge (Table 1). Addition of protein on the surface of AgNPs was further confirmed qualitatively by TEM (Figure 1A). Modifications in spectra of AgNPs were found following addition of PCs leading to a spectral red shift (spectral shift to the right) (Figure 1B). Incubation of AgNPs with proteins for 8 h was found to influence the dissolution of AgNPs as assessed by ICP-MS (Figure 2). Specifically, addition of HSA and BSA was found to reduce AgNP dissolution, whereas addition of HDL increased dissolution compared with AgNPs in water. Such an observation may be rationalized in terms of the net reduction in ζ-potential of AgNP-HDL compared with other samples (Table 1), which indicates that HDL displaced citrate groups and destabilized AgNPs resulting in increased dissolution.

TABLE 1.

Characterization of AgNP With and Without PC in Water

Particle Hydrodynamic size (nm) ζ–Potential (mV)
AgNP 19.13 ± 1.95 −35
AgNP-HSA 69.99 ± 3.03 −25
AgNP-BSA 30.60 ± 0.08 −18
AgNP-HDL 62.10 ± 1.79 −8
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FIG. 1.

FIG. 1.

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Protein corona (PC) formation on the surface of 20 nm citrate-suspended AgNPs. A, Representative transmission electron microscopy (TEM) images of AgNPs with individual PCs of human serum albumin (HSA), bovine serum albumin (BSA), and high-density lipoprotein (HDL). B, Hyperspectral spectral analysis demonstrating a red shift in spectra due to the addition of individual PCs on 20 nm AgNPs.

FIG. 2.

FIG. 2.

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Dissolution of 20 nm citrate-suspended AgNPs. AgNPs were incubated for 8 h in the presence of water (control), human serum albumin (HSA), bovine serum albumin (BSA), or high-density lipoprotein (HDL). Following incubation, AgNPs were centrifuged for 10 min at 14 000 rpm (20 817 g) and the supernatant was collected for inductively coupled plasma-mass spectrometry (ICP-MS) analysis of Ag+ content. Values are expressed as mean of triplicate ± SD.

Alterations in protein structure were evaluated by circular dichroism spectroscopy after incubation with AgNPs as compared with the pure protein structure (values appearing in bold) (Table 2). Association of proteins with AgNPs was found to decrease the number of α-helices in all PCs, likely due to entropy-driven protein unfolding and the formation of hydrogen bonding between the proteins and the AgNPs, except for AgNP-HDL where the number of α-helices increased (Table 2). HDL differs from BSA and HSA in that it possesses lipid content and a greater degree of structural complexity and heterogeneity. The number of β-sheets was found to increase in all proteins upon association with AgNPs. Protein turns were found to increase in AgNP-HSA and AgNP-HDL while being reduced in AgNP-BSA. The number of unordered proteins was increased in AgNP-HSA and AgNP-BSA while being reduced in AgNP-HDL.

TABLE 2.

Changes in Protein Structure Upon Association with AgNPs

Protein Structure HSA AgNP-HSA BSA AgNP-BSA HDL AgNP-HDL
α-Helices 52 48 45 41 31 33
β-Sheets 14 15 14 19 14 15
Turns 6 7 16 14 17 21
Unordered 27 31 25 27 39 30
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Influence of AgNP-Protein Corona on Cellular Uptake

To assess differences in cellular uptake due to the addition of the PC, cells were exposed for 2 h to 25 µg/ml of particles with or without the PC and assessed for changes in side scatter shift by flow cytometry (Figure 3A) and ICP-MS (Figure 3B). Increases in side scatter shift are indicative of increased cellular granulation and therefore provide a qualitative assessment of particle uptake. Due to RAEC and RLE cells primarily expressing SR-BI compared with other scavenger receptors, we addressed its role in the AgNP uptake and toxicity (Dieudonne et al., 2012; Uittenbogaard et al., 2000). To investigate the role of SR-BI in mediating the uptake of these particles, cells were treated with an SR-BI inhibitor (Blt2) for 30 min prior to exposure. All cells demonstrated particle uptake following exposure to AgNPs as shown by increased side scatter shift compared with serum-free media controls (SF) (Figure 3A—normalized uptake in RLE and RAEC cells). The addition of the PC (HSA, BSA, or HDL) to AgNPs was found to reduce particle uptake in both cell types in comparison with uptake of AgNPs without a PC (Figure 3A). Inhibition of the SR-BI receptor was shown to diminish side scatter shift to control levels after exposure to AgNPs indicative of diminished uptake and/or cell-nanoparticle interactions (Figure 3A). Changes in SSC due to the addition of the PCs were further validated by ICP-MS demonstrating that the addition of the PC reduced AgNP uptake compared with AgNPs without a PC in both cell types (Figure 3B). Enhanced dark field microscopy also qualitatively demonstrated the reduction of uptake following the addition of the PC and the role of SR-BI (Figure 4). In an attempt to understand how changes in the characteristics of the AgNPs due to the addition of the PC influence cellular internalization, uptake measured by changes in side scatter shift and ICP-MS were individually correlated with both hydrodynamic size (Supplementary Figure S1) and ζ-potential (Supplementary Figure S2). No strong correlations however were determined between hydrodynamic size and uptake (Supplementary Figure S1). There was a correlation between ζ-potential and uptake as measured by ICP-MS for RLE (R2 = 0.6164) and RAEC (R2 = 0.7905) (Supplemental Figure 2). Uptake as measured by changes in side scatter shift (SSC) and ICP-MS were found to be in agreement and to correlate for RLE (R2 = 0.8608) and RAEC (R2 = 0.9032) (Supplementary Figure S3).

FIG. 3.

FIG. 3.

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Measurement of AgNP uptake by assessment of changes in mean side scatter shift (SSC) via flow cytometry and inductively coupled plasma-mass spectrometry (ICP-MS). A, Rat lung epithelial cells (RLE), or rat aortic endothelial cells (RAEC) were either pretreated for 30 min with serum-free media without the scavenger receptor-BI (SR-BI) inhibitor (Blt2) or with Blt2 (50 μM). Cells were exposed to AgNP, AgNP-HSA, AgNP-BSA, or AgNP-HDL at a concentration of 25 μg/ml for 2 h and assessed for changes in side scatter shift (SSC). SSC values of cells exposed to AgNPs with or without protein coronas (PCs) were normalized to serum-free media-treated control cells to produce a fold change. Values are expressed as mean ± SEM (n = 4–10 per group). Asterisk indicates significant difference from AgNPs without PC (P < .05). B, RLE and RAEC were pretreated with serum-free media or Blt-2 (50 μM) and subsequently exposed to AgNPs without or with PCs (25 μg/ml) for 2 h. Cells were then assessed by ICP-MS for changes in uptake. Values are expressed as mean ± SEM (n = 3 per group). Asterisk indicates significant difference from AgNPs without a PC (P < .05).

FIG. 4.

FIG. 4.

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Representative enhanced dark field images of rat lung epithelial cell (RLE) (A), and rat aortic endothelial cells (RAEC) (B) pretreated with serum-free media with or without the scavenger receptor-BI inhibitor (Blt2) for 30 min and then exposed for 2 h to AgNP, AgNP-HSA, AgNP-BSA, or AgNP-HDL (25 μg/ml).

Intracellular Modifications to the AgNP-Protein Corona

Following a 2 h exposure to AgNPs with or without PCs at 25 µg/ml, hyperspectral analysis was performed on intracellular AgNPs and compared with their original spectra (Figure 5). Following RLE and RAEC internalization, spectra from AgNPs with PCs underwent a blue shift (a shift to the left) and were similar to AgNPs without a PC suggesting intracellular loss of the PC. The spectra of AgNPs without a PC however were not found to be considerably altered compared with their original spectra.

FIG. 5.

FIG. 5.

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Differences in hyperspectral profiles of internalized AgNPs without and with individual protein coronas (PCs) compared with original samples. A, Rat lung epithelial cells (RLE), and B, rat aortic endothelial cells (RAEC) were exposed for 2 h to 25 μg/ml of AgNP, AgNP-HSA, AgNP-BSA, or AgNP-HDL on microscope chamber slides and fixed with paraformaldehyde. Cells were assessed by Cytoviva hyperspectral dark field microscopy selecting a minimum of 1000 pixels of AgNPs and comparing the generated mean spectrum to the original sample. Numbers represent the wavelength of the spectral peak.

Role of Protein Corona and SR-BI in AgNP-Induced Cytotoxicity

In general, all AgNPs (with or without PCs) demonstrated a dose-dependent reduction in viability in both cell types at 3 and 6 h following exposure (Figure 6). Addition of all PCs to AgNPs was found to reduce cytotoxicity in both RLE and RAEC at a concentration of 50 µg/ml at both 3 and 6 h (Figure 6). In addition, RAEC exposed to AgNPs with a HDL PC at a concentration of 25 µg/ml was also shown to reduce cytotoxicity at 3 and 6 h (Figure 6). To determine the role of scavenger receptor-B in the toxicity induced by AgNP, cytotoxicity was evaluated in RLE and RAEC cells following treatment with a SR-BI inhibitor (Blt2). Following pretreatment with Blt2 (50 µM), cells were assessed for cytotoxicity 3 h postexposure to 50 µg/ml AgNPs with or without PCs. This 3 h time point and the concentration of 50 µg/ml were selected as significant differences were found between groups. Treatment with Blt2 (50 µM) alone was not found to cause cytotoxicity in either cell type (data not shown). Similar to Figure 5, addition of the PCs was found to reduce AgNP-induced cytotoxicity in both RLE and RAEC cell types (Figure 7A). Further inhibition of SR-BI was found to reduce cytotoxicity induced by exposure to AgNPs with or without PCs in RAEC cells (Figure 7A).

FIG. 6.

FIG. 6.

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Cell viability changes in rat lung epithelial cells (RLE) (A), and rat aortic endothelial cells (RAEC) (B) at 3 and 6 h following exposure to AgNP, AgNP-HSA, AgNP-BSA, or AgNP-HDL. Cells were exposed to 6.25, 12.5, 25, or 50 μg/ml AgNPs with or without various protein coronas. Values are expressed as mean ± SEM (n = 3–6 per group). Asterisk indicates significant difference from AgNPs without a PC at the same exposure concentration, suspension, and time point (P < .05).

FIG. 7.

FIG. 7.

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Cytotoxicity in rat lung epithelial cells (RLE) (A) and rat aortic endothelial cells (RAEC) (B) at 3 h following exposure to AgNP, AgNP-HSA, AgNP-BSA, or AgNP-HDL with or without a scavenger receptor-B inhibitor (Blt2). Cells were exposed to 50 μg/ml AgNPs with or without various protein coronas (PCs) following pretreatment with either in serum-free media or serum-free media with Blt2 (50 µM). Cell activation in RLE and RAEC (B) was reduced following inhibition of scavenger receptor B. Gene expression of IL-6 was determined via real-time PCR analysis after a 1 h exposure to 50 μg/ml of AgNP, AgNP-HSA, AgNP-BSA, or AgNP-HDL following pretreatment with serum-free media with or without the scavenger receptor-B inhibitor (Blt2) (50μM). Values are expressed as mean ± SEM (n = 3–6 per group). Asterisk indicates significant difference from the corresponding AgNPs without the antagonist Blt2 present (P < .05). Hash symbol indicates significant difference from AgNPs without PC (P < .05).

Influence of Protein Corona on Cell Activation by AgNPs

Cell activation was assessed through measuring the induction of IL-6 mRNA following a 1 h exposure to 50 µg/ml of AgNPs or AgNPs with a PC. All AgNPs were found to induce IL-6 expression in the two cell types (Figure 7B). Pretreatment with Blt2 alone was not found to induce IL-6 expression (Figure 7B). In RLE cells, exposure to AgNP-HDL was shown to induce a greater IL-6 expression, compared with AgNP without a PC (Figure 7B). Inhibition of SR-BI in RLE cells was shown to inhibit AgNP-induced IL-6 expression in all groups (Figure 7B). RAECs exposed to AgNPs without the addition of a PC were found to induce more IL-6 expression compared with AgNPs with a PC (HSA, BSA, and HDL) (Figure 7B). Pretreatment of RAECs with Blt2 was found to inhibit AgNP-induced IL-6 expression in all groups (Figure 7B). To understand the effect of uptake on the activation of cells, mRNA expression of IL-6 was correlated with uptake as measured by changes in side scatter shift and ICP-MS (Supplemental Figure 4). IL-6 mRNA expression was determined to correlate with RAEC uptake of AgNPs with and without the PC as measured by changes in SSC (R2 = 0.9869) and ICP-MS (R2 = 0.9048); however, no correlation was determined between uptake and IL-6 mRNA expression of RLE (Supplementary Figure S4).

DISCUSSION

Association of proteins on the surface of NPs can modify their therapeutic applications and toxicity by altering their uptake and distribution. In this study we addressed the role of the protein corona in the toxicity induced by AgNPs in two cell types that are common targets of NP exposure (epithelial and endothelial cells). Common proteins found in human serum (HSA and HDL) and in cell culture media (BSA) were found to associate with AgNPs. Specifically, albumin and HDL have been found to readily accumulate on the surface of carbon-based NPs and AgNP following incubation in fetal bovine serum (Shannahan et al., 2013a, b). Association of these individual proteins was found to modify the physicochemical properties of the AgNPs, as well as induce conformational changes in protein structure. The addition of the PC was found to influence cellular uptake of AgNPs and affect both cytotoxicity and cellular inflammatory responses. Finally, SR-BI was determined to mediate the uptake of AgNPs and the corresponding inflammatory activation and cytotoxicity.

AgNPs were found to readily associate with the proteins utilized in this study, likely through hydrogen bonding and electrostatic and hydrophobic interactions. This association not only modified the interface of the AgNPs to affect their cell entry and ion release but also caused conformational changes in individual protein structure. It is likely that these conformationally modified proteins, adsorbed onto or released from the surface of the AgNP, could facilitate inflammatory responses and/or autoimmune reactions due to their altered structure and compromised biofunctionality.

Overall, AgNPs without the PC were found to be readily taken up and to initiate cytotoxicity and an inflammatory response by both RLE and RAEC. Interestingly, the addition of the PC to AgNPs was found to decrease uptake, and at higher concentrations (25 and 50 µg/ml) reduced cytotoxicity. Addition of HSA and BSA PCs were determined to decrease the inflammatory response in both RLE and RAEC cell types, whereas addition of the HDL PC was found to enhance the inflammatory response in RLE cells. These decreases in uptake and cellular responses due to the PC may be influenced by the AgNP suspension. A previous study utilizing TEM to assess uptake of AgNPs in HEK cells has demonstrated similar responses as seen in our study in terms of citrate-suspended AgNP uptake related to suspension material (Monteiro-Riviere et al., 2013). Specifically, the association of IgG on 20 nm citrate-suspended AgNPs reduced HEK cell uptake, whereas association of IgG on 20 nm silica-suspended AgNPs enhanced uptake (Monteiro-Riviere et al., 2013). The decrease in uptake and cellular response may be influenced by alterations in surface charge and reactivity due to formation of the PC. Importantly, these findings illustrate differences in PC formation, which may alter the reactivity of AgNPs thereby modulating uptake, and subsequent cellular responses. Thus, the type of initial AgNP suspensions (i.e., citrate, polyvinylpyrrolidone, or silica stabilized) may influence the PC formation based on ligand exchange/competition and influence uptake and subsequent cellular responses. Therefore, examining the PC formation as a function of AgNP suspension material requires further study.

Research has demonstrated that AgNPs upon entry into the body undergo various transformations causing the release of Ag+ (Liu et al., 2012). Upon uptake into cells the release of Ag+ is increased compared with water-suspended AgNPs (Singh and Ramarao, 2012). This increase in dissolution is likely due to various intracellular mechanisms including changes in pH, oxidative interactions, and/or complexation with intracellular proteins, especially those with thiol groups (Liu et al., 2012; Singh and Ramarao, 2012). It is currently unclear how the addition of the PC may influence mechanisms of dissolution of AgNPs both extracellularly and intracellularly. In this study, addition of albumin PCs was found to decrease AgNP dissolution extracellularly. The addition of an albumin PC may stabilize AgNPs thereby decreasing dissolution. It is also likely that more dynamic interactions with proteins resulting in constant interchanges may enhance AgNP dissolution as seen with HDL. The rate of NP-protein exchange is likely determined by numerous NP physicochemical properties and protein characteristics making it a very complex system to model, particularly in the presence of multiple proteins. Thus, further study assessing the role of the PC on dissolution rates of AgNPs is needed to fully understand the role of Ag+ in the toxicity induced by AgNP exposure. It is likely that the PC, through modulating cell surface receptor recognition and uptake will also alter the intracellular amount of AgNPs and thereby influence the amount of intracellular Ag+ release and toxicity.

These differences in uptake after the addition of the PC may be attributed to various physicochemical parameters such as variations in surface charge, length, shape, chemical composition, stability of polymer coating, abundance of protein accumulation, PC-induced particle aggregation, and/or suspension material displacement by proteins. In our study AgNPs were shown to have a negative ζ-potential (citrate is a negatively charged suspension material), however, when coated with the proteins used in this study their surface charge drops drastically due to charge neutralization. With this decrease in surface charge there is a corresponding decrease in cellular uptake of AgNPs. It is likely that these modifications in surface charge alter the intracellular uptake mechanisms mediated by membrane fluidity due to AgNP partitioning and electrostatic and/or hydrophobic interactions between AgNP-PC and membrane proteins, thereby contributing to differences in AgNP internalization. Interestingly, although having the least surface charge, PCs consisting of HDL were found to have similar uptake to AgNPs without a PC. This uptake of AgNP-HDL is likely driven by the SR-BI receptor’s affinity for HDL, as well as the high lipid content of the HDL for initiating partitioning with the cell membranes. The SR-BI receptor is also known to mediate the uptake of not only HDL but negatively charged materials in general. Treatment with an inhibitor of SR-BI was found to diminish intracellular uptake by epithelial and endothelial cells further supporting the role of surface charge in uptake mechanisms. It is unclear in our study how the abundance of proteins, which associated with the AgNPs forming a PC-affected uptake. These possible differences in protein accumulation may also modify particle aggregation in suspension and alter the uptake of AgNPs. It is likely that a citrate coating enhances NP uptake and that the addition of proteins displaced citrate molecules on the surface causing a decrease in uptake. Further study is necessary to understand the mechanisms by which citrate influences the formation of the PC and its role in facilitating intracellular uptake. Additionally, this study only addresses a simplified PC, however, in vivo and in cell culture a complex PC will form which may influence cellular responses. The formation of the PC will likely be extremely diverse not only between different NPs but also due to variations in biological media.

The observation of red-shifts in the spectra of protein-coated AgNPs (compared with uncoated pristine AgNPs) in the hyperspectral analysis confirmed the initial loading of our selected proteins (see red curves in Figure 5) on the surfaces of AgNPs (Podila et al., 2012). However, upon internalization, the protein-coated AgNP spectra exhibited a loss of this red-shift suggesting a loss of the PC. Interestingly, as evident from Figure 5, the protein-coated AgNPs showed an additional blue-shift in addition to the loss of red-shift (i.e., the internalized protein-coated AgNPs, blue curves in Figure 5, exhibit a spectral peak <611 nm). Such an exacerbated shift may possibly arise from: (1) increased intracellular dissolution occurring as the PC is being removed internally, (2) removal of the citrate coating, and/or (3) replacement of the original protein coating by new cellular proteins. Notably, our data also support the influence of the PC on cell surface receptor recognition resulting in a varied cellular uptake of AgNPs and ensuing activation. Together our results suggest that the PC may influence cell surface receptor-mediated signaling and uptake leading to differential intracellular dosimetry and differences in toxicity.

In summary, our study demonstrates that the formation of the PC can alter cellular uptake and responses through the SR-BI receptor. These findings have implications in the fields of nanotoxicology and nanotherapeutics. Our results demonstrate that the addition of the PC could influence nano-sized drug targeting through either enhancing or decreasing cellular uptake and clearance, which could alter interactions with other cell types and their in vivo biodistribution or in vitro dosimetry in cellular models. Furthermore, nanotherapeutics, that specifically target the SR-BI receptor, may be beneficial through increasing uptake; however, activation of scavenger receptors may exacerbate inflammatory responses. In conclusion, the PC is a complex and dynamic entity. Nevertheless, an understanding of its biological consequences is necessary for the development of safe nanomedicines and allaying concerns regarding unintended toxicity. Furthermore, our results highlight the need for in vitro toxicity assessment of nanomaterials to be performed in various cell culture medias and nanomaterial suspensions to understand how the formation of the PC may influence biological responses. Due to the complexity of the PC, comprehensive computational modeling approaches will be needed to understand not only PC formation, evolution and dynamics, but also its cellular interactions.

SUPPLEMENTARY DATA

Supplementary data are available online at http://toxsci.oxfordjournals.org/.

FUNDING

NIEHS (grants R01 ES019311 and U19 ES019525; as well as the NIEHS Centers for Nanotechnology Health Implications Research [NCNHIR]).

Supplementary Material

Supplementary Data
supp_143_1_136__index.html (1.1KB, html)

ACKNOWLEDGMENTS

Any opinions, findings, conclusions, or recommendations expressed herein are those of the authors and do not necessarily reflect the views of the National Institute of Environmental Health Sciences/NIH. The authors have no conflicts of interest to disclose.

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