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. 2023 Jan 10;8(3):3310–3318. doi: 10.1021/acsomega.2c06882

Silver Nanoparticle Surface Chemistry Determines Interactions with Human Serum Albumin and Cytotoxic Responses in Human Liver Cells

Kira M Fahy †, Madeline K Eiken †,‡, Karl V Baumgartner †,‡, Kaitlyn Q Leung †, Sarah E Anderson †, Erik Berggren †, Evangelia Bouzos †,‡, Lauren R Schmitt †, Prashanth Asuri ‡,*, Korin E Wheeler †,*
PMCID: PMC9878656  PMID: 36713725

Abstract

graphic file with name ao2c06882_0007.jpg

Engineered nanomaterials (ENMs) are synthesized with a diversity of surface chemistries that mediate biochemical interactions and physiological response to the particles. In this work, silver engineered nanomaterials (AgENMs) are used to evaluate the role of surface charge in protein interactions and cellular cytotoxicity. The most abundant protein in blood, human serum albumin (HSA), was interacted with 40 nm AgENMs with a range of surface-charged coatings: positively charged branched polyethyleneimine (bPEI), negatively charged citrate (CIT), and circumneutral poly(ethylene glycol) (PEG). HSA adsorption to AgENMs was monitored by UV–vis spectroscopy and dynamic light scattering, while changes to the protein structure were evaluated with circular dichroism spectroscopy. Binding affinity for citrate-coated AgENMs and HSA is largest among the three AgENM coatings; yet, HSA lost the most secondary structure upon interaction with bPEI-coated AgENMs compared to the other two coatings. HSA increased AgENM oxidative dissolution across all particle types, with the greatest dissolution for citrate-coated AgENMs. Results indicate that surface coating is an important consideration in transformation of both the particle and protein upon interaction. To connect results to cellular outcomes, we also performed cytotoxicity experiments with HepG2 cells across all three AgENM types with and without HSA. Results show that bPEI-coated AgENMs cause the greatest loss of cell viability, both with and without inclusion of HSA with the AgENMs. Thus, surface coatings on AgENMs alter both biophysical interactions with proteins and particle cytotoxicity. Within this study set, positively charged bPEI-coated AgENMs cause the greatest disruption to HSA structure and cell viability.

I. Introduction

Because of the antimicrobial properties of silver engineered nanomaterials (AgENMs), they are increasingly used in consumer and medicinal products.1,2 Their wide application includes markets such as food packaging, active wear, and cosmetics.3 The unique and advantageous properties of AgENMs have also led to their increased use in biomedical products, such as diagnostic tools,4 biosensors,5 and wound dressings.6 With their prevalence of use, AgENMs are also one of the most widely studied ENMs. Yet, control over AgENM interactions in biological systems remains elusive. A complete understanding of the biological interactions of AgENMs requires fundamental knowledge of how AgENMs interact with biomolecules.

When ENMs enter into a biological system, a protein corona rapidly forms on the surface.7,8 This adsorption of proteins and other biomolecules gives the ENMs a new biological identity, altering fate and transport.9−15 For AgENMs, this protein corona can serve as more than a passive surface coating. The biocidal activity of AgENMs is largely due to oxidative dissolution, formation, and release of Ag(I)(aq), disrupting biomolecular structure and function. Metal-binding proteins, in particular, catalyze the formation and release of Ag(I)(aq) from AgENMs. Downstream, bactericidal effects are due to the disruption of key enzymes,16 lipid membranes,17−19 and reactive oxygen species (ROS) generation.20,21 Although there are minimal concerns about toxicity to eukaryotes, the subtle effects from repeated minimal exposure scenarios are rare, with the exception of a few long-term, low-level exposure studies. Because of the large surface area to volume ratio of nanomaterials, a significant number of proteins or other biomolecules can interact with AgENMs to mediate their biological interactions. Understanding and controlling the formation and reactivity of the AgENM protein corona is the first step toward a better understanding of downstream cellular response.22,23

While the core composition of the ENM mediates the protein corona formation to some extent, the tunable surface coating surrounding the ENM serves as the interface with which proteins interact.24−28 The ENM surface coating not only functions to stabilize the particle but can also be modified to target biochemical interactions.28 Variants in surface coating, like zwitterionic coatings, can be used to decrease formation of the corona.29,30 Other changes, such as varying coating charge, can alter the strength of protein interactions and the resulting protein structure.31−35 The ENM surface not only modifies the formation of the protein corona but can also alter cellular uptake of ENMs and toxicity;23,36 however, contradictory data exists on the influence of protein corona on AgENM uptake and toxicity.37−40 Despite a variety of efforts to assess the role of surface coating in mediating protein interactions and subsequent cellular response, additional fundamental studies are required to develop a framework for predicting how surface coating will alter both molecular-level interactions with proteins and cellular-level response.

In this study, we focus on three common AgENM surface coatings and assess their interactions with a model protein, human serum albumin (HSA). As the most abundant protein in blood, HSA has already been well characterized in studies of gold ENMs, but studies have yet to focus on HSA interactions with AgENMs of varied surface coatings. HSA was allowed to interact with a small library of 40 nm AgENMs with surface coatings of varied charges, including positively charged branched polyethyleneimine (bPEI), negatively charged citrate (CIT), and circumneutral poly(ethene glycol) (PEG) coatings. This model system provides a baseline understanding of the biophysicochemical interactions that take place at the ENM–protein interface, including changes to the size and surface chemistry of the AgENM, alterations to the protein structure, and agglomeration of AgENM–protein complexes. AgENM toxicity was evaluated as a result of the physical changes at the AgENM surface. Knowing how the surface chemistries of ENMs can mediate their behavior in biological systems will give rise to the engineering of safer and more effective therapies utilizing nanotechnology.

II. Experimental Section

II.I. Materials

Sodium citrate monobasic, sodium phosphate, hydrochloric acid, and HSA were purchased from Sigma-Aldrich (St. Louis, MO). BioPure 40 nm Ag nanospheres with bPEI, CIT, and PEG surface coatings were ordered from nanoComposix (San Diego, CA). All experiments were conducted using nanopure water (18.2 MΩ at 25 °C). Human hepatoma (HepG2) cells were obtained from ATCC (Manassas, VA), Dulbecco’s modified Eagle medium (DMEM) from Mediatech (Manassas, VA), fetal bovine serum and penicillin–streptomycin from Invitrogen (Carlsbad, CA), sodium pyruvate and minimum essential media (MEM) nonessential amino acids from Life Technologies (Carlsbad, CA), trypsin/ethylenediaminetetraacetic acid (EDTA) from Cellgro (Manassas, VA), and WST cell proliferation assay kit from Dojindo Molecular Technologies (Rockville, MD).

Stock solutions of HSA were prepared to a concentration of 5 M, aliquoted, and frozen at −20 °C for later use. Because the various techniques used across this study have different sample concentration requirements, the concentration of HSA and AgENMs was varied to enable measurements and improve the signal-to-noise ratio for each experiment. However, the molar ratio of HSA to AgENMs was kept constant except for the inductively coupled plasma mass spectrometry (ICP-MS) experiments. The ratio was chosen to estimate a monolayer AgENM coating if all HSA were to bind, as estimated in previous studies.41,42

II.II. Circular Dichroism (CD) Spectroscopy

For CD analysis, protein dilutions were prepared at 0.0625 mg/mL HSA in a 5 mM sodium citrate buffer, pH 6.5. For samples with AgENMs, the HSA was incubated with 0.00625 mg/mL AgENM in a total volume of 280 μL for roughly 24 h. Scans were taken in a quartz cylindrical cuvette with a path length of 1 mm and analyzed with an Olis Rapid-Scanning Monochromator. The wavelength range recorded was 185–260 nm, and the number of increments set to 150. The percent α helix of the protein was calculated using eq 1 43 and the measured ellipticity ([θ]) at 208 nm. Ellipticity in deg·cm2/dmol was calculated using eq 2,44 where Cp is the concentration of protein in mg/mL and l is the path length in mm.

II.II. 1
II.II. 2

II.III. UV–Vis Spectroscopy

Samples were prepared in a 5 mM sodium citrate buffer of pH 6.5 with 0.004 mg/mL AgENM. HSA with concentrations ranging from 0 to 500 nM was titrated in and the 10 samples were left to incubate at room temperature for roughly 24 h. A high-resolution spectrum of each 650 μL of sample was collected using disposable plastic cuvettes with a Shimadzu UV–Vis 3600. Each titration is an average of three trials. Binding constants, Ka, were determined by fitting the data to a Langmuir adsorption isotherm,45eq 3

II.III. 3

HSA was titrated into AgENMs to obtain the change in wavelength, Δ, and maximum change in wavelength, Δmax, at various concentrations of HSA, [HSA].

II.IV. Hydrodynamic Diameter and ζ-Potential Characterization

For Z-average hydrodynamic diameter and ζ-potential measurements (ZetaPlus from Brookhaven Laboratories), 5 μg/mL HSA and 0.55 μg/mL AgENMs were incubated for roughly 24 h, then filtered with a 0.02 μm inorganic membrane filter (Whatman). A Smoluchowski model was used to calculate ζ-potential from electrophoretic mobility measurements.

II.V. Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

Samples for inductively coupled plasma mass spectrometry (ICP-MS) were prepared in chelex-treated nanopure water. For 6 h, 0.06 mg/mL AgENMs were reacted with 40 mg/mL HSA. Samples were then centrifuged (30 min, 21,000g) to remove ENMs. The supernatant was removed and recentrifuged, then this final supernatant was analyzed for silver ion (Ag(I)) concentration. Ag(I) concentration was determined using an Agilent 7500CE ICP-MS (Agilent Technologies, Palo Alto, CA) by the Interdisciplinary Center for Plasma Mass Spectrometry (University of California at Davis, CA). The samples were introduced using a MicroMist Nebulizer (Glass Expansion, Pocasset, MA) into a temperature-controlled spray chamber. Instrument standards diluted from Certiprep 2A (SPEX CertiPrep, Metuchen, NJ) encompassed the range 0, 0.5, 1, 10, 50, 100, 200, 500, and 1000 parts per billion (ppb) in 3% trace element grade HNO3 (Fisher Scientific, Fair Lawn, NJ) in nanopure water. A separate 100 ppb Certiprep 2A standard was analyzed as every tenth sample as a quality control. Sc, Y, and Bi Certiprep standards (SPEX CertiPrep) were diluted to 100 ppb in 3% HNO3 and introduced by peripump as an internal standard.

II.VI. Cell Culture and Viability Assessment

HepG2 cells were maintained in DMEM supplemented with 10% fetal bovine serum, sodium pyruvate, MEM nonessential amino acids, and 1% penicillin–streptomycin at 37 °C in a 5% CO2 humidified environment. They were grown in 100 mm tissue culture dishes (Greiner Bio-One, Monroe, CA) and passaged every 7–10 days using 0.25% trypsin/EDTA. For the cell viability assay, cells were first seeded in 96-well flat-bottom plates (15,000 cells per well) and allowed to proliferate until confluence (48 h). Before exposing the cells to various AgENM preparations, HSA-coated AgENMs for the cell viability assays were prepared by reacting 20 μg/mL with 0.467 mg/mL HSA or 2 μg/mL with 0.0467 mg/mL HSA for 10 min in DMEM. Cells were then washed with culture media and treated with either culture media alone or culture media containing 2 or 20 μg/mL of bare or HSA-coated AgENMs in triplicate (200 μL total volume per well) and incubated for three days at 37 °C in a humidified atmosphere containing 5% CO2, before collection on days 1 and 3. At collection, the cells were washed with media to remove the AgENMs, and cell viability was measured using the formazan-based WST assay as per the manufacturer’s instructions. Briefly, WST solution (20 μL) was added to culture media (200 μL), followed by a 2 h incubation period at 37 °C in a humidified atmosphere containing 5% CO2. Absorbance was measured at 570 nm using a Tecan Infinite 200 PRO plate reader (Tecan, Switzerland). Background absorbance due to AgENMs was recorded using no-cell controls for all of the AgENM preparations and subtracted from the absorbance values of the experimental samples. A protein-only control was also performed using 0.467 mg/mL HSA or 0.0467 mg/mL HSA to confirm that any excess HSA did not lead to a measurable toxicity. Viable cell percentages were thereafter calculated relative to controls without AgENM treatment. Cell viability assays were also similarly carried out following treatment with culture media containing 0.024, 0.24, or 2.4 μg/mL silver nitrate (AgNO3) to assess the cytotoxic effects of dissolved silver ions.

III. Results and Discussion

Several techniques were utilized in this study to characterize the biophysicochemical transformations that occur to both the protein of interest, HSA, and the AgENM, 40 nm silver nanospheres. Dynamic light scattering (DLS) and ζ-potential gave insights into the agglomeration state and surface charge, respectively, of AgENMs and AgENM–HSA complexes. Circular dichroism (CD) was used to observe protein secondary structural changes of the particle upon adsorption to the AgENM. Particle–protein binding affinity was calculated by constructing adsorption isotherms from surface plasmon resonance data collected by UV–vis spectroscopy. Finally, insights into AgENM toxicity were explored through ICP-MS data of dissolved silver ions and cell viability assays.

Although every effort was made to harmonize conditions across techniques used across molecular-level characterizations and cellular-level studies, each have different condition requirements.

III.I. HSA Effects on AgENM Agglomeration and Surface Charge

To assess the role of HSA in mediating AgENM transformations in aqueous solution, dynamic light scattering and ζ-potential were employed to determine hydrodynamic diameter and surface charge.35,46 As shown by a hydrodynamic diameter of 40–60 nm, all three AgENMs are stable in water. The higher polydispersity for AgENM(CIT) and (bPEI) is indicative of a widened size distribution (Figure S1). Upon addition of HSA, the hydrodynamic diameter of bPEI- and PEG-coated particles increased modestly, but the hydrodynamic diameter of the CIT-coated AgENMs nearly tripled (Table 1). Although the mechanism of agglomeration is not clear, the adsorption isotherm studies suggest there is steady binding of protein on the AgENM surface leading to agglomeration of HSA–AgENM(CIT) complexes. We, along with previous studies,33,43,47 suggest that the change in hydrodynamic diameter is due to electrostatic interaction between the particle and the protein. Given that the protein has positively and negatively charged patches and interacts with particles of different charged surfaces, there is an interaction that increases the overall hydrodynamic diameter.

Table 1. DLS and ζ-Potential Characterization of AgENMs with and without HSAa.

  AgENMs (bPEI) AgENMs (bPEI) + HSA AgENMs (CIT) AgENMs (CIT) + HSA AgENMs (PEG) AgENMs (PEG) + HSA
size (nm) 55.6 ± 3.6 71.2 ± 8.7 42.2 ± 2.9 118.8 ± 7.6 57.2 ± 3.8 64.8 ± 2.5
polydispersity 0.204 ± 0.034 0.226 ± 0.062 0.316 ± 0.017 0.260 ± 0.079 0.197 ± 0.014 0.208 ± 0.027
ζ-potential (mV) 4.4 ± 2.0 –2.1 ± 1.8 –29.9 ± 2.5 –17.3 ± 0.47 –2.1 ± 0.8 –11.4 ± 1.8
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a

Error bars represent standard deviation of three samples.

For ζ-potential, it is not surprising that bPEI-coated particles are most positive, PEG-coated are circumneutral, and CIT-coated are negative. Upon addition of HSA, the ζ-potential of bPEI- and PEG-coated particles decreased, resulting in slightly negative potentials. Conversely, CIT-coated particles became more positive after HSA addition (Table 1). However, all ENM–HSA coronas exhibited an overall negative surface charge, similar to outside studies.46,48 The change in ζ-potential indicates that HSA is adsorbing to the ENM surface to an extent, depending on the ENM coating, given that the net electrostatic charge of HSA is about −16 mV under experimental conditions. Again, the addition of HSA resulted in electrostatic interactions between the protein and the ENM. The surface charge was evaluated with the sorption of protein on the AgENMs and the formation of a complex. Since CIT-coated AgENMs have the highest detectable binding and highest change in hydrodynamic diameter, it is not surprising that the ζ-potential of the CIT-coated AgENMs and HSA complex changed the most. The overall surface charge of the complex reflects the surface charge of the protein, suggesting HSA coats the CIT-coated particles. Due to weaker and undetectable binding with the bPEI- and PEG-coated ENMs, the surface charge reflects the solution as a whole, with its protein and particle substituents.

III.II. Protein Stability upon Interaction with AgENMs

As proteins adsorb to the surface of the ENMs, conformational changes in the protein structure have the potential to alter the identity of the protein corona.49 Protein deformability alters not only the function of the protein but also the ability of other proteins to adsorb to the ENM surface and the possibility for a biological response to the ENM–protein complex. Changes in the protein secondary structure upon addition of AgENMs in this system were probed with CD spectroscopy. CD spectra of the proteins and protein–AgENM solutions are shown in Figure 1.

Figure 1.

Figure 1

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Representative CD spectra demonstrating the effect of AgENMs on the secondary structure of HSA. CD spectra were obtained at an HSA concentration of 0.065 mg/mL and AgENM concentrations of 0.0065 mg/mL in 5 mM sodium citrate buffer pH 6.5. Spectra were smoothed using a 15-point Savitzky–Golay filter. Spectra shown are representative of three or more replicates.

The HSA structure changed slightly upon the interaction with each of the three AgENMs, as indicated by changes in the secondary structure observed in CD spectra. Based on these spectra, the percent α-helical content of HSA was calculated for each sample (Table 2). Although PEG-coated AgENMs caused only a 2% loss in the α-helical content, bPEI- and CIT-coated AgENMs reduced the α-helical content by 8.6 and 6.2%, respectively. A similar unfolding driven by surface charge was noted in Dennison et al. where BSA deformed more upon interaction with positively coated AuENMs compared to a negatively charged CIT-coated AuENM. We note that all of the proteins, bound and unbound, contribute to the CD signal.50 Thus, if we assume that that HSA that is not bound to ENMs is structurally intact, the reduction in the secondary structure calculated here may underrepresent structural disruption for HSA at the ENM surface.

Table 2. Percent Change in Ellipticity upon Treatment of HSA with AgENMsa.

coating % α helix change (%)
control (no ENM) 52.6 ± 5.4%  
bPEI 44.0 ± 7.2% –8.6
CIT 46.4 ± 4.8% –6.2
PEG 50.6 ± 2.8% –2.0
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a

Error bars represent standard deviation of three samples.

III.III. AgENM–Protein Binding Affinities

Taking advantage of the plasmonic properties of AgENMs, plasmon peak shifts were monitored to assess binding affinity with increasing protein concentrations in solution. A red shift indicates a change in the refractive index of the ENM complex, suggesting protein is bound to the ENM surface.51 The adsorption of HSA to AgENMs with bPEI, CIT, and PEG coatings was tested by observing the shift in wavelength at maximum absorbance (Figure 2). The resulting data was fit to the Langmuir isotherm, which has been used in other protein–ENM studies.31,33,47 Fit results generated a Ka of 2.08 × 107 M–1 (R2 = 0.97). The Langmuir isotherm includes several assumptions, including homogeneous binding of single or submonolayer coverage, reversible binding, and full equilibration on the experimental time scale.31,45

Figure 2.

Figure 2

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Langmuir adsorption isotherm of CIT-coated AgENMs with HSA at concentrations from 0 to 500 nM. Isotherms were recorded using UV–vis spectroscopy after equilibrating various concentrations of HSA with 0.004 mg/mL AgENMs in 5 mM sodium citrate at pH 6.5. Error bars represent standard deviation of three samples, and the R-squared value for the Langmuir fit was 0.969.

The association constant for HSA and CIT-coated AgENMs is consistent with previous reports. Riley et al., for example, report an association constant that is within error of ours (2.2 ± 0.3 × 107 M–1)52 for BSA and AgENM(CIT). Unfortunately, there are few other AgENM studies to point to for a direct comparison, but AuENMs have a broader body of literature and can provide comparative trends. Boulos et al. found that a wide range of association constants have been reported for studies using different methods to obtain association constants for BSA- and CIT-coated AuENM interactions; association constants range from about 103 to 1011 M–1.31 This suggests that methods and binding conditions are important considerations when assessing results.

Unfortunately, under these solution conditions, the shifts in wavelength as HSA was titrated into solutions of bPEI- and PEG-coated AgENMs were too small to measure, indicating that this technique is not sensitive enough to detect the low binding of HSA to bPEI- and PEG-coated AgENMs. This result is consistent with other studies showing that BSA binds more strongly to CIT-coated AuENMs than to positively and PEG-coated particles.33 The apparent lack of binding to the positively charged particle is also supported by Dennison et al.,33 whereby at the relative concentration of protein to ENM, the LSPR technique employed is not sensitive enough to detect the low binding.

The stronger interaction between HSA and CIT-coated AgENMs could be understood through the electrostatic surface of the HSA molecule. Though HSA has an overall negative charge, the surface of the protein contains different regions of positive and negative charge, as seen in Figure 3. It is possible that HSA is oriented in a way that presents positively charged patches that interact strongly with the CIT-coated AgENM surface, but electrostatically repel the bPEI-coated AgENMs. The idea that proteins bind to ENMs with preferential binding regions, or orientations, is established in other systems. A recent report from Tollefson et al. used a complementary experimental and computational approach to determine the binding interface between cytochrome c and AuENMs; although binding is neither rigid nor fully specific, there are clear regions of preferential binding.53

Figure 3.

Figure 3

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Surface presentation of electrostatic potential for HSA. Positively charged regions of the molecular surface are shown in blue and negatively charged regions in red. The structure presentations were generated using PDBID 4K2C in PyMOL 2.4.0.

III.IV. HSA-Driven AgENM Oxidative Dissolution

Previous studies have demonstrated that proteins can increase AgENM oxidation and dissolution to form Ag+(aq).54−58 Because this dissolved silver cation is key to the biocidal activity of AgENMs, we used ICP-MS to monitor the role of HSA in catalyzing the formation of Ag+(aq) (Figure 4). Since kinetic studies have shown that the protein-driven dissolution is most rapid in the first 4 h, the 6 h end point here enabled us to measure maximum dissolution across samples.52 Also, Boehmler et al. have shown that while dissolution occurs at lower protein concentrations, it is measurably lessened.52 Therefore, we performed the ICP-MS experiments using a higher HSA/AgENM ratio than our other studies reported here to measurably compare the effects of AgENM coatings on oxidative dissolution. Although some oxidative dissolution of AgENMs occurs naturally, there was a dramatic increase in Ag+(aq) formation upon addition of HSA. The greatest increase in oxidative dissolution occurred in the CIT-coated AgENMs, followed by PEG and bPEI coatings. The larger dissolution for CIT-coated AgENMs can be explained, in part, because this coating is the most labile, enabling easier access to the core silver. This is consistent with previous studies, where the protein enhances dissolution of AgENMs, presumably due to uptake of the oxidized Ag(I)(aq) by the exposed thiols in the proteins.52,57,59−62

Figure 4.

Figure 4

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AgENM dissolution in the presence of HSA and media. Amount of silver in the AgENM samples that oxidized and dissolved to form Ag(I)(aq) was measured using ICP-MS after reacting 0.06 mg/mL AgENM with 40 mg/mL for 6 h. Error bars represent standard deviation of three samples, and statistical significance was independently evaluated for each AgENM sample in HSA and media relative to the corresponding sample in water using a two-tailed t-test evaluated at the 95% (*) or 99% (**) confidence interval.

HSA likely mediates oxidative dissolution of AgENMs through metal-binding amino acids that take-up oxidized silver at the ENM surface.56 Based upon hard–soft acid–base theory, these metal-binding amino acids are most likely cysteine and methionine, but density functional theory calculations also suggest that arginine, lysine, and histidine could strongly bind Ag+.63 HSA is proposed to enhance the oxidative dissolution of AgENMs via coordination of chemisorbed Ag(I)(aq) to thiol groups present within the protein.59,60,62 This nucleophilic dissolution is dependent on the concentration of the protein in solution42 and is enhanced through the displacement of Ag(I)-coordinated proteins by other proteins in solution.56,57,62 The rate and extent of dissolution is further complexed by the dependence on size, shape, and curvature of the AgENM. For example, Riley et al. demonstrate that the rate of dissolution of AgENMs is dependent upon both nanoparticle size and concentration of BSA.52 The results presented also show the role of surface coating on AgENM dissolution.

III.V. Cell Viability

Given that the applications of ENMs are expanding into new areas that may directly or indirectly impact human health,1,64,65 we also investigated the effects of protein coatings on the responses of human cells to AgENMs. Human liver hepatoma HepG2 cells were chosen as the model cell line because they have been widely used to determine in vitro toxicity of various chemical and nanomaterial preparations.66−70 Furthermore, several articles have published that the liver is a target organ for several nanomaterials, due to their accumulation in the liver after ingestion, inhalation, or absorption.71−73 To quantify the role of HSA–AgENM interactions on cell viability, HepG2 cells were incubated with low (2 μg/mL) and high (20 μg/mL) concentrations of bare and HSA-coated AgENMs for three days (concentrations selected based on previous literature and preliminary experiments in our lab that suggested that the silver nanoparticle IC50 is ca. 1–10 μg/mL).74 WST assay, a tetrazolium salt-based commercially available metabolic assay, was used to study the responses of HepG2 cells to bare and protein-coated nanoparticles. The data revealed that the cell viability was strongly influenced by AgENM concentration and surface chemistry (Figure 5). Consistent with previous literature,75−77 our data indicated that bPEI-coated particles were significantly more toxic than CIT- and PEG-coated particles. Also in line with previous research, we observed that the protein coating had a significant impact on the cellular responses to the AgENMs, at least for AgENMs with bPEI and CIT coatings. No significant differences in cell viability were observed for PEG AgENMs coated or not coated with HSA. Interestingly, we observed that the HSA coating led to an increase in toxicity of CIT-coated AgENMs, but a decrease in toxicity of bPEI-coated particles. While a decrease in toxicity of AgENMs upon exposure to proteins is a well-reported phenomenon,38,78,79 increased toxicity of protein-coated ENMs relative to bare ENMs is not a consistently reported finding. However, Barbalinardo et al. demonstrate that the adsorption of serum proteins onto the surface of CIT-coated AgENMs is essential for the uptake and cytotoxicity of AgENMs in mouse embryonic fibroblasts,40 which is consistent with our results where CIT-coated AgENMs complexed with HSA have reduced cell viability relative to “bare” CIT-coated AgENMs.

Figure 5.

Figure 5

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Percent cell viability of HepG2 cells exposed to AgENMs. Cell viability was determined by WST assay after exposing cells to 2 μg/mL (left) and 20 μg/mL (right) bare and HSA-coated AgENMs on day 1 (light bars) and day 3 (dark bars). Cell viability data was corrected by subtracting background absorbance due to nanoparticles and normalized against no AgENM controls. Error bars represent standard deviation of three samples, and statistical significance was independently evaluated for each AgENM–HSA condition relative to the no protein control using a two-tailed t-test evaluated at the 95% (*) or 99% (**) confidence interval.

The observed differences in the effects of protein coatings on the toxicity of different AgENMs are also consistent with the changes in ζ-potential of AgENMs upon addition of HSA. For instance, the observed increase in ζ-potential of CIT-coated AgENMs may lead to increased cellular uptake of the ENM–protein complex,80,81 thereby leading to an increase in toxicity.80 This hypothesis is consistent with the literature that suggests the differences in ENM-mediated toxicity can be assigned to one or a combination of attributes that contribute to cell toxicity, including cellular binding and uptake.82 Recent research using ENM–protein complexes prepared using cationic and anionic particles has also reported that cell binding and receptor targeting are independent of ENM type or size, but strongly dependent on ENM charge. These observations indicate that differences in the structure of adsorbed proteins can translate into differences in cellular binding and internalization.50 Further mechanistic insights connecting molecular changes at the surface of ENMs to cellular response or toxicity will require new tools that enable researchers to follow the path of protein–ENM complexes into the cell.

Toxicity of AgENM can also be partly explained by the release of Ag(I)(aq) ions;83−85 any differences in the release of ionic Ag(I) caused by the protein coatings can present as differences in cell toxicity. We tested if the oxidative dissolution of AgENMs mediated by HSA (as shown in Figure 4) led to differences in cell viability. We exposed HepG2 cells to Ag(I) salts spanning the concentration range 0.024–2.4 μg/mL (low (2%) and high (12%) bounds of HSA-driven AgENM oxidative dissolution for 2 μg/mL and 20 μg/mL AgENM concentrations). However, we did not observe any significant toxicity for the silver salts; cell viability was greater than 80% for all of the tested Ag(I) salt concentrations (Figure S2). Collectively, these studies indicate that the observed changes in cell viability were primarily dependent on AgENM toxicity and cannot solely be explained by HSA-catalyzed release of ionic silver.

IV. Summary and Conclusions

In this work, we reaffirm the importance of oxidative dissolution and changes in engineered coatings in mediating the toxicity of AgENMs. We also connect these changes in toxicity to protein-driven dissolution and changes in AgENM surface properties. Of the coatings examined here, CIT-coated AgENMs bind HSA most tightly and showed the greatest dissolution in the presence of HSA. Yet, positively charged, bPEI-coated AgENMs resulted in the greatest loss of HSA secondary structure and demonstrated the highest toxicity. This implies that engineered surface coatings are a central consideration in AgENM toxicity, even in the presence of a protein corona. These results highlight an interplay between AgENM dissolution and the role of AgENM surface-coating-mediated interactions in the mechanisms of AgENM toxicity and cellular response. Further study is needed to evaluate the complete molecular mechanisms of AgENM interactions with the cell, as well as the role of more complex protein coronas in biomediated AgENM transformations.

Acknowledgments

Support for this work came from the National Institute of Environmental Health Sciences of the National Institutes of Health (R15ES025929). K.F. is grateful for support from the Jean Dreyfus Lectureship for Undergraduate Institutions. M.E., S.A., and E.B. thank the Clare Boothe Luce program. E.B. is also grateful for Kuehler Undergraduate Research Scholar funds. K.B. and L.S. received funding from SCU as a Hayes Scholar and Bastiani Scholar programs, respectively. The table of contents figure was created with BioRender.com.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.2c06882.

  • Representative DLS histograms of the AgENMs with and without HSA (Figure S1) and percent cell viability of HepG2 cells after exposing them to various concentrations of AgNO3 (Figure S2) (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao2c06882_si_001.pdf (389.3KB, pdf)

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