Influence of cysteine and bovine serum albumin on silver nanoparticle stability, dissolution, and toxicity to Phanerochaete chrysosporium

Abstract

The transport and environmental persistence of silver nanoparticles (AgNPs) after exposure to dissolved organic matter (DOM) are influenced by their stability and dissolution. In addition, their toxicity to microorganisms is influenced by the release of silver ions (Ag⁺). Here, we characterize the stability and dissolution of citrate-stabilized AgNPs, and their toxicity to a white-rot fungus, Phanerochaete chrysosporium (P. chrysosporium), following exposure to either cysteine (CYS) or bovine serum albumin (BSA). The results indicated that both CYS and BSA changed the diameter, zeta potential, and Ag⁺ dissolution of AgNPs. Bacterial viability and intracellular reactive oxygen species (ROS) levels were investigated to determine the toxicity of AgNPs to P. chrysosporium. In this study, CYS decreased the inhibitory effects of 100 μg L⁻¹ Ag⁺ and 10 mg L⁻¹ AgNPs in a concentration-dependent manner. At higher concentrations of CYS, the toxicity of Ag⁺ and AgNPs was reduced dramatically. However, cell viability decreased at higher BSA concentrations (≥50 mg L⁻¹), suggesting a more complex reaction than simply binding to the released Ag⁺. Addition of 10 mg L⁻¹ AgNPs significantly stimulated ROS production, but the addition of CYS and BSA decreased the ROS level in a concentration-dependent manner. In summary, our results provide useful information in understanding the fate, transformation, and toxicity of citrate-stabilized AgNPs in the natural environment.

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1. Introduction

Silver nanoparticles (AgNPs; 1–100 nm in size) with specific mechanical, optical, electronic, and catalytic properties, are widely used in consumer and medical products.^1,2 Recent studies have shown that AgNPs released to the natural environment undergo profound chemical transformations that can affect their stability, bioavailability, and toxicity. Indeed, the release of AgNPs into the environment has a severe effect on numerous organisms, including microbial communities, fungi, algae, plants, vertebrates, invertebrates, and human cells.³ The bactericidal activity of AgNPs is attributable to the controlled release of silver ions (Ag⁺) and nano-specific effects of AgNPs that can be internalized by the bacterial capsule and bind to proteins, enzymes, and DNA, disrupting normal cell function and even causing cell death.^4–7 The mechanisms that induce toxicity of AgNPs may be related to the production of reactive oxygen species (ROS). In some cases, AgNPs are more toxic than equivalent concentrations of silver salts. Ionic silver may occur in AgNP suspensions, both as residuals from particle synthesis and from particle oxidation. The stability of AgNPs in the environment significantly affects their toxicity to organisms. However, little is known about the transport, fate, and toxicity of AgNPs in natural environments. A wide range of environmental parameters is predicted to influence the stability of AgNPs, as well as their dissolution in aqueous solutions, including pH, ionic strength, and the presence of dissolved organic matter (DOM). Therefore, it is essential to identify the material and environmental parameters that control the behavior and environmental impact of AgNPs.

DOM is ubiquitous in natural water, but the interaction between AgNPs and DOM is not well-understood. During the release of AgNPs into aquatic environments, DOM plays an important role in altering the surface charge of AgNPs, and thus their stability. Several studies have demonstrated that DOM, such as fulvic and humic acids, reduces the aggregation of citrate-stabilized AgNPs.^8–10 The interaction of AgNPs with DOM in the environment can have dramatic effects on their dissolution rate, stability, and toxicity. Therefore, a clear understanding of DOM–AgNP interactions is essential to elucidate the toxicity and stability of AgNPs in the aquatic environment. Different types of DOM have different structures and compositions that can affect the mobility, bioavailability, and toxicity of AgNPs and release of Ag⁺. Several studies observed that the presence of DOM reduced the toxicity of AgNPs to some model organisms.^11–13 Lan et al. concluded that sulfur- and nitrogen-rich DOM significantly increased colloidal stability.¹⁴

The objective of this study was to investigate the effect of different types of DOM on the stability of AgNPs in water, and to understand the toxic effects of AgNPs in the presence of DOM. Here, cysteine (CYS) and bovine serum albumin (BSA) were selected as the two different types of DOM. Phanerochaete chrysosporium (white-rot fungi) was selected as the model organism, as it has been widely used to treat waste water streams containing heavy metals and toxic organic pollutant.^15,16 CYS is a strong, silver ligand, known for its affinity to complex with free Ag⁺.^17–19 Several studies have demonstrated that CYS decreases AgNP dissolution, and attenuates the toxicity of AgNPs.^20,21 The presence of proteins within the solution can stabilize the AgNPs against aggregation.²² Other studies observed that BSA reduced the toxicity of AgNPs to Nitrosomonas europaea by chelating the released Ag⁺, and further reduced toxicity by binding to the surface of the AgNPs.¹⁹ CYS and BSA are two different types of dissolved organic matter (DOM), they are ubiquitous in natural water and both CYS and BSA are thiol-containing biomolecules. They can decrease the toxicity of AgNPs and influence the aggregation of AgNPs. Previous studies found they can decrease AgNPs dissolution and toxicity, but the mechanism is not very clear. Therefore, we chose the CYS and BSA as two different types of dissolved organic matter (DOM) and compared their differences between them. Thus, it is important to investigate the interaction between DOM and AgNPs to evaluate the transformation and cytotoxicity of AgNPs in the environment.

2. Materials and methods

2.1. Synthesis and characterization of AgNPs

Citrate-stabilized AgNPs were synthesized following our previously reported method.² Briefly, an ice bath with 59.8 mL of a solution containing 0.6 mM trisodium citrate and 0.4 mM NaBH₄ was prepared in ultrapure water and stirred vigorously. We then introduced 0.24 mL of 50.0 mM AgNO₃, after which the solution turned yellow. The solution was then stirred again for a period of 3 h at room temperature, at a stirring rate of 540 rpm. Following synthesis, citrate-stabilized AgNPs were concentrated into a stock solution using centrifugal ultrafiltration (Amicon Ultra-15 3K, Millipore, USA). The concentration of the stock solution was monitored by flame atomic absorption spectrometry (AAS700, PerkinElmer, USA). A 30 mg L⁻¹ AgNP stock solution was stored at 4 °C before use. The morphology of citrate coated AgNPs was characterized by transmission electron microscope on a JEOL JEM-3010 (JEOL, Japan) at 120 kV. TEM samples were prepared by placing a drop of stock AgNPs suspension onto a carbon-coated copper grid and dried at room temperature overnight and each sample gets three photographs and analyzed by Gatan Digital Micrograph 3.9. The localized surface plasmon resonance (LSPR) spectra were obtained using a UV-vis light spectrophotometer with a wavelength from 300 to 650 nm (Model UV-2550, Shimadzu, Japan). The hydrodynamic diameter and zeta (ζ) potential of AgNPs were determined by dynamic light scattering (DLS) using a nanoseries zetasizer (Malvern Instruments) with a 633 nm laser source and a detection angle of 173°.

2.2. AgNPs dissolution

Due to the localized surface plasmon resonance (LSPR) of AgNPs, and the corresponding UV-vis absorption peak is sensitive to particle size, shape, aggregation state.²³ Thus, UV-vis spectroscopy is a widely used method for characterizing the aggregation state of AgNPs suspensions and can quantify the AgNPs in solution and thus, quantify the amount of dissolution that has occurred over a given time period.^24–26 According to previous study, the full width at half-maximum (FWHM) was calculated from LSPR adsorption spectrum to determine if the decrease in the sample's λ_max intensity was influenced by aggregation in addition to dissolution.¹⁹ In dissolution experiments, 5 mg L⁻¹ AgNPs was present while the concentrations of CYS and BSA varied from 0.1 to 100, 1 to 500 mg L⁻¹, respectively. CYS and BSA were purchased from Aladdin. The LSPR were measured at five time points, immediately before the addition of CYS/BSA to the centrifuge tube (t = 0), 3 h after the addition of CYS/BSA (t = 3 h) and 6 h, 9 h, and 12 h after addition of CYS/BSA (t = 6 h, t = 9 h and t = 12 h). AgNPs dissolution was quantified by measuring the intensity change of λ_max over time, as A. K. Ostermeyer et al. previously described, and comparing λ_max intensity of the sample against the λ_max intensity of an AgNPs standard curve ranging from 0 to 10 mg L⁻¹. The percentage of dissolution was calculated using the following formula:

% dissolution = (1 − [AgNPs]_{t=12 h}/[5 mg L⁻¹ AgNPs])

Concentrations of Ag⁺ in suspensions were determined by flame atomic absorption spectrometry graphite furnace (AAS). The samples were first centrifuged with an ultrafiltration centrifuge (Amicon® Ultra-4 3K, Millipore, USA), and then the filtrate was digested using ultrapure HNO₃. The digested samples were used to determine Ag⁺ using AAS.

2.3. Biotoxicity assay

2.3.1. Fungal culture. The white-rot fungal strain P. chrysosporium (BKMF-1767) was obtained from the Chinese Typical Culture Center of Wuhan University. The fungus was cultivated in Kirk inorganic liquid medium in Erlenmeyer flasks (500 mL) and incubated at 150 rpm for 48 h at 37 °C. The mycelia were harvested by centrifugation and three washes of a 2 mM sodium bicarbonate buffer solution. Equal amounts of mycelia (0.2 g) were added to test tubes, and tests were performed using several freshly prepared concentrations of different mixes of DOM and AgNP solutions. After 12 h, test tubes were centrifuged to collect the supernatant.

2.3.2. Cell viability. Cell viability was assessed using the MTT assay, according to Chen et al.²⁷ 0.2 g P. chrysosporium pellets were mixed with 1 mL MTT solution (5 g L⁻¹) and incubated for 2 h at 50 °C. MTT were obtained from Sigma-Aldrich. The reaction was suspended by adding 0.5 mL hydrochloric acid (1 M) to the mixture. The mixture was then centrifuged (10 000 × g, 5 min) to collect the supernatant, and the pellets agitated in 6 mL propan-2-ol for 2.5 h. The centrifugation process was repeated and the absorbency of the supernatant was recorded as 534 nm.

2.3.3. Intracellular ROS assay. Intracellular ROS generation was determined by monitoring the intensity of fluorescence of the different exposed samples using a fluorometric indicator. Bacterial samples were incubated with 5 μM 2′7′-dichlorodihydrofluorescein diacetate (DCFH-DA) for 2 h before they were exposed to the DOM and AgNP-mixed solution. DCFH-DA were obtained from Sigma-Aldrich. The medium then was removed, and the cells were washed with phosphate-buffered saline (PBS). The DCFH-DA passively enters into the cell where it reacts with ROS to form the highly fluorescent compound dichlorofluorescein (DCF), which indicated the extent of ROS generation. The quantitative analysis of intracellular ROS level was detected using fluorescence spectrometer (FluoroMax-4; Horiba Scientific, Tokyo, Japan) with filters for excitation at 485 nm and emission at 525 nm. Thus, enhanced fluorescence is indicative of an elevated level of ROS in the cell. Similarly, for the qualitative analysis of intracellular ROS by fluorescence microscopy, the cells were treated and processed as indicated above. Finally, after washing with PBS, the stained cells were mounted onto a microscope slide in mounting medium and were observed by a Laser Scanning Confocal Microscope (LSCM) (Olympus TY1318, Japan).

3. Results and discussion

3.1. Characteristics of citrate-stabilized AgNPs

Synthesized citrate-stabilized AgNPs were found to be relatively monodispersive in size, with an average diameter of 20.63 ± 15.16 (Fig. S1A†). The pH value of AgNPs suspension was 6.1. In situ, DLS gave an average diameter of 23.27 ± 0.62 nm, which was larger than that determined by TEM. Comparing the sizes evaluated from DLS and TEM, the notable difference in particle diameter can be ascribed to the preparation of sample. The size measured by DLS corresponded to the core and the swollen corona of the micelles, whereas TEM images depicted the core for micelles at the dried state of sample, and hence AgNPs exhibited a larger DLS size.²⁸ The discrepancy is likely due to the formation of AgNPs aggregates. To verify the formation of the prepared AgNP suspension, the LSPR peaks are presented in the UV-vis absorbance spectra, the peak wavelength of the AgNPs was 397 nm (Fig. S1B†). Size distribution by volume of citrate-stabilized AgNPs (Fig. S1C†), indicate that the AgNPs in water suspensions were monodispersed. The AgNPs carried a strong, negative charge with a zeta potential of −35.95 ± 0.98 mV in ultrapure water. A typical batch of AgNPs contained 30 mg L⁻¹ total silver, of which 12 μg L⁻¹ was residual Ag⁺ (aq). The stock solution was yellow (Fig. S1D†) and stored at 4 °C for two months. Before each experience period we have measured the concentration of Ag⁺, the result shows the ionic concentration changed little (≤20 μg L⁻¹) and was almost negligible.

3.2. Impact of CYS and BSA on AgNP stability

The measures of DLS diameter, zeta potential, and the LSPR spectra were used to characterize the stability of AgNPs in the aqueous media.^29–31 Using the DLS and UV-vis methods, the data collected for DLS diameter and zeta potential were then compared with the measurable modifications in the LSPR spectra to assess the potential changes in particle stability in the presence of CYS and BSA.

The DLS measurements revealed a slight decrease in DLS average diameter and increase in average zeta potential with increasing CYS concentrations (Fig. 1A). The decrease in the diameter of AgNPs with exposure to CYS was potentially caused by the formation of smaller AgNPs. TEM measurements (Fig. S2†) obtained after addition CYS to AgNPs suspension. The sizes of these AgNPs (14.64 ± 11.67) are smaller than that of the initial AgNPs. According to previous study that the CYS can induce AgNPs disintegration and form smaller AgNPs. Mechanistically, the smaller AgNPs in the AgNPs/CYS solutions can be formed through two pathways. The first is by etching, where the individual AgNPs are gradually etched into smaller AgNPs. The second is via the decomposition pathway in which the Ag⁺–CYS is converted into small AgNPs that contain zero valence silver atoms together with the capping thiolate.³² The zeta potential of AgNPs increased from −35.95 ± 0.98 to −27.90 ± 0.71 mV with increasing CYS concentrations. The increase in zeta potential could be due to the exchange of trivalent citrate anions with monovalent CYS anions on the surface of the AgNPs. CYS is capable of adsorbing onto AgNPs and thus can affect the surface charge, reactivity, and stability of the AgNPs, which subsequently could influence the interaction of AgNPs with metal contaminants. Upon the addition of 0.1 mg L⁻¹ CYS to AgNP solution, the LSPR band red-shifted toward a longer wavelength. However, the absence of a further, significant shift in LSPR band of AgNPs with subsequent addition of CYS clearly suggested that the adsorbed CYS acted as a good capping agent, as it prevented the individual AgNPs from aggregation. Moreover, CYS significantly decreased citrate-stabilized AgNPs LSPR absorbance in a concentration-dependent manner (Fig. 2A). Furthermore, relatively lower LSPR absorbance of citrate-stabilized AgNPs at higher CYS concentrations suggests adequate cysteine-coating of the surface of AgNPs, as reported by Liu et al.³³

Fig. 1 Effects of (A) CYS, and (B) BSA on the average diameter and zeta potential of citrate-stabilized AgNPs.

Fig. 2 (A) Changes in the localized LSPR spectra of citrate-stabilized AgNPs upon amendment with variable concentrations of CYS; [CYS]: (a) 0, (b) 0.1, (c) 1, (d) 10, and (e) 100 mg L⁻¹. (B) Changes in the localized LSPR spectra of citrate-stabilized AgNPs upon amendment with variable concentrations of BSA; [BSA]: (a) 0, (b) 1, (c) 10, (d) 100, and (e) 200 mg L⁻¹.

As shown in Fig. 1B, the average diameter of AgNPs increased from an initial value of 23.11 ± 0.53 to 29.47 ± 0.76 nm with increasing BSA concentrations, with the exception of the two lower concentrations, 1 and 5 mg L⁻¹. The slight decrease in average diameter at lower BSA concentrations could be due to the formation of smaller AgNPs induced by BSA. The increased average diameter is may attributed to BSA (a good capping agent and can substitute weakly bound capping agents and with an average molecular weight of 66 430 Da) coated to the surface of AgNPs and substitute citrate. Previously, BSA was shown to attach to nanoparticle surfaces, forming a monolayer of approximately 7 nm thickness.^34,35

The zeta potential of AgNPs increased from −35.95 ± 0.98 to −9.25 ± 1.06 mV with increasing concentrations of BSA, suggesting that an interaction between BSA and the surfaces of the AgNPs. Citrate–AgNPs have a negative zeta potential (−35.95 ± 0.98), however, this zeta potential can be modified using other organic coatings. The zeta potential of AgNPs increased from −35.95 ± 0.98 to −9.25 ± 1.06 mV with increasing concentrations of BSA is the results of surface change of AgNPs after addition of BSA. The absorption spectra of AgNPs in the presence of increasing concentrations of BSA are shown in Fig. 2B. Upon the addition of increasing concentration of BSA to AgNPs solution, the LSPR band of red-shifted toward a longer wavelength with a slight decrease in its absorption intensity. The slight red shift in LSPR and little change in λ_max suggests that the BSA interacted with AgNPs, and dissolution of AgNPs were occurred. The discrepancy of Fig. 1B and 2B can be ascribed to the different characterization measurement (DLS vs. UV-vis). The change of average diameter and zeta potential resulted from interaction between BSA and the surfaces of the AgNPs. However, the UV-vis method is only related to localized surface plasmon resonance (LSPR) of AgNPs core, while DLS measure the AgNPs including shell. BSA induced shell variation caused obvious DLS data change.

3.3. Impact of CYS and BSA on AgNP dissolution

AgNPs are well-known to exhibit strong LSPR, and the corresponding UV-vis peak is sensitive to particle size, shape, and aggregation state. LSPR has proven a very useful technique for the analysis of the transformation of AgNP suspension.^36–38 Unlike other methods, the UV-vis absorption spectroscopy method can be used to measure the amount of silver remaining in AgNP form, even in complex solutions. Our results show that optical absorption at the height of the LSPR peak is proportional to total AgNP concentration (Fig. S3†). The UV-vis absorption of the AgNP–CYS solution exhibited a decrease in the LSPR signal at ∼397 nm over time (Fig. 3). AgNPs in test media containing CYS resulted in an increased FWHM (Table S1†) and the decrease in λ_max may be due to the creation of AgNPs aggregates as well as AgNPs dissolution. AgNPs in test media containing 0.1, 10, 50, and 100 mg L⁻¹ CYS resulted in 28.8%, 48.5%, 49.3%, and 50.8% decreases in AgNP concentration, respectively, after 12 h (Fig. 3, Table 1) Thus, the presence of CYS reduced the AgNP concentration due to the AgNPs aggregation as well as AgNPs dissolution. The FWHM after addition BSA with a constant (Table S2†) indicate that the AgNPs suspension was not aggregating and that decreases in λ_max are due to dissolution. As BSA concentrations increased from 1 to 10, 100, and 500 mg L⁻¹, the AgNP concentration decreased by 9.5%, 15.4%, 23.7%, and 39%, respectively, after 12 h (Fig. 4, Table 2). Therefore, the presence of BSA reduced the AgNP concentration due to AgNPs dissolution.

Fig. 3 UV-vis absorption spectra of 5 mg L⁻¹ AgNPs after addition of CYS at (A) 0.1 mg L⁻¹, (B) 10 mg L⁻¹, (C) 50 mg L⁻¹, and (D) 100 mg L⁻¹.

Table 1 Concentration (mg L⁻¹) of AgNPs in the presence of CYS

CYS concentration	0 h	3 h	6 h	9 h	12 h
0.1 mg L^−1	4.17 ± 0.48	3.95 ± 0.23	3.68 ± 0.55	3.59 ± 0.22	3.56 ± 0.20
10 mg L^−1	3.77 ± 0.75	3.28 ± 0.33	2.87 ± 0.47	2.70 ± 0.82	2.58 ± 0.61
50 mg L^−1	3.71 ± 0.83	3.18 ± 0.28	2.85 ± 0.76	2.69 ± 0.10	2.53 ± 0.88
100 mg L^−1	3.61 ± 0.27	3.06 ± 0.34	2.79 ± 0.77	2.62 ± 0.51	2.46 ± 0.24

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Fig. 4 UV-vis absorption spectra of 5 mg L⁻¹ AgNPs after addition of BSA at (A) 1 mg L⁻¹, (B) 10 mg L⁻¹, (C) 100 mg L⁻¹, and (D) 500 mg L⁻¹.

Table 2 Concentration (mg L⁻¹) of AgNPs in the presence of BSA

BSA concentration	0 h	3 h	6 h	9 h	12 h
1 mg L^−1	4.69 ± 0.12	4.59 ± 0.81	4.59 ± 0.52	4.57 ± 0.92	4.52 ± 0.14
10 mg L^−1	4.67 ± 0.74	4.40 ± 0.11	4.33 ± 0.28	4.28 ± 0.75	4.23 ± 0.36
100 mg L^−1	4.60 ± 1.01	4.20 ± 0.78	4.40 ± 0.13	3.99 ± 0.42	3.82 ± 0.86
500 mg L^−1	4.21 ± 0.52	3.60 ± 0.33	3.41 ± 0.47	3.33 ± 0.77	3.05 ± 0.12

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Fig. 5 shows the dissolution of Ag⁺ from citrate-stabilized AgNPs in the presence of various concentrations of CYS and BSA over time. In the absence of CYS and BSA, the AgNPs dissolved slowly over time. An initial increase in dissolved Ag⁺ concentration was observed within 6 h after the addition of CYS, which then subsequently decreased. In this study, the addition of CYS, at all concentrations, resulted in lower dissolved Ag⁺ concentrations after 9 h than in the absence of CYS. This effect may have been due to the reduction of Ag⁺ back to Ag⁰ in the presence of CYS.²¹ Previous studies noted that CYS increased the dissolution of Ag and slowed aggregation of AgNPs for chelate dissolved Ag⁺.^39,40 The addition of BSA (0–500 mg L⁻¹) decreased the concentration of Ag⁺ over time. Our results contrast with a previous study by Ostermeyer et al.¹⁹ that reported that BSA chelated the Ag⁺ released from the AgNPs, and increased AgNP dissolution. These contradictory results may be due to the differences in experimental methodology. In addition, BSA is capable of forming polymeric species.⁴¹ Thus, the ultrafiltration membranes used in this study may have made it possible to retain significant amounts of dissolved Ag in certain conditions.

Fig. 5 Dissolved Ag⁺ from citrate-stabilized AgNPs (total Ag = 5 mg L⁻¹) in the presence of various (A) CYS, and (B) BSA concentrations as a function of time over 12 h. Error bars are sample standard deviations from triplicate measurements.

3.4. Impact of CYS and BSA on AgNP toxicity to P. chrysosporium

Previous studies suggested that the toxicity of AgNPs to microorganisms can be attributed to the Ag⁺ released from AgNPs.⁴² A recent report suggested that while AgNPs and Ag⁺ may have similar effects on bacterial survival, they have distinct mechanisms of antibacterial activity.⁴³ Exposure of P. chrysosporium cells to AgNPs and Ag⁺ led to significant reductions in cell viability, depending on the type of silver, concentration, and exposure time. Concentration-dependent viability of P. chrysosporium was observed for both the citrate-stabilized AgNPs and free Ag⁺ (as added AgNO₃; Fig. S4†). Cell viability clearly decreased with increasing Ag⁺ concentration. However, the cell viability fluctuations with increasing concentrations of both AgNPs and Ag⁺ together indicated higher toxicity than that of AgNPs alone. The positive effect of citrate-stabilized AgNPs on cell viability can be attributed to the overcompensatory behavior of microorganisms. Previous studies have reported that sublethal concentrations of silver may enhance bacterial fitness and hinder antimicrobial applications.^42,44 Our results indicated that the toxicity induced by Ag⁺ and AgNPs was a concentration-dependent effect. In this study, we chose 10 mg L⁻¹ AgNPs and an equivalent amount of Ag⁺ (8 μg L⁻¹) as is dissolved in the 10 mg L⁻¹ AgNPs to be an experimental concentration for the following experiments which it was a moderate concentration for cytotoxicity.

Fig. 6 provides insight into how CYS and BSA affect toxicity of Ag⁺ and AgNPs to P. chrysosporium. In this study, CYS decreased the inhibitory effects of 8 μg L⁻¹ Ag⁺ and 10 mg L⁻¹ AgNPs in a concentration-dependent manner. At higher concentrations of CYS, the toxicity of Ag⁺ and AgNPs was reduced dramatically, suggesting that CYS was a suitable ligand to decrease Ag⁺ and AgNP availability. The addition of BSA to the culture medium resulted in enhanced cell viability in the presence of Ag⁺ and AgNPs. Our results shows that the CYS and BSA have little influence on fungus viability and intracellular ROS level (see Fig. S5†). It is thought that the silver-induced cell toxicity was mainly due to the release of Ag⁺. Fig. 6 also shows that AgNPs is more toxic than an equivalent amount of Ag⁺ as is dissolved in the AgNPs. Although the toxicity of AgNPs is partly explained by the release of Ag⁺, the specific nanoparticle effect still remains. After entering cell the AgNPs also can release Ag⁺ to damage fungus. Previous studies suggested that BSA can reduce AgNP inhibition through two potential mechanisms: (1) bind to Ag⁺ by ligand groups within the BSA molecule, including thiol and CYS; and (2) coat the AgNPs to prevent direct interactions with the bacteria.¹⁹ Interestingly, however, cell viability decreased at higher BSA concentrations (≥50 mg L⁻¹), suggesting a more complex reaction than simply binding to released Ag⁺. This result may be due to the interaction between Ag⁺–BSA complexes and P. chrysosporium. Metal ion–albumin complexes are rapidly internalized by cells.⁴⁵ Our results imply that the effect of BSA on the toxicity of Ag⁺ and AgNPs are not easily predicted by either the magnitude of the Ag⁺–BSA complex, or the extent of BSA association with the surfaces of the AgNPs.

Fig. 6 Effect of (A) CYS, and (B) BSA on the viability of P. chrysosporium exposure to AgNPs and as an equivalent amount Ag⁺ released form AgNPs, respectively. Error bars are sample standard deviations from triplicate measurements. Experimental conditions: AgNP concentration = 10 mg L⁻¹; Ag⁺ concentration = 8 μg L⁻¹; exposure time = 12 h.

Oxidative stress is one of the critical mechanisms of cytotoxicity induced by AgNPs.^46–48 The intracellular ROS will perturb the redox potential equilibrium, causing an intracellular pro-oxidant environment and ultimately result in the disruption of cell functions. To investigate the potential roles of CYS and BSA in influencing oxidative stress induced by AgNPs, we measured intracellular ROS generation by DCFH-DA assay using spectrofluorometry and confocal microscopy. Fig. 7A and B shows that 10 mg L⁻¹ AgNPs could significantly stimulate ROS production. However, with the addition of CYS and BSA, the ROS level decreased in a concentration-dependent manner. This pattern was also confirmed by confocal microscopy images (Fig. 7C–K), showing an obvious decrease in the ROS level with the addition of CYS or BSA. These results suggest that CYS and BSA decreased ROS levels induced by AgNPs. Previous studies have shown that CYS can simultaneously act as complexing, nucleophilic, reducing, and protecting agents.³⁵ CYS can coat the surfaces of AgNPs and chelate the Ag⁺ to form CYS–Ag⁺ complexes. BSA protected cells from inhibition by AgNPs by utilizing the dual mechanisms of binding to the Ag⁺ with thiol groups and coating the surfaces of the AgNPs, thus reducing the interactions between cells and AgNPs.

Fig. 7 Effect of (A) CYS and (B) BSA on the intracellular ROS generation of P. chrysosporium following exposure to 10 mg L⁻¹ AgNPs. Detection of hyphae ROS level using confocal microscopy (C) negative control, (D) positive control, (E) 10 mg L⁻¹ AgNPs, (F–H) CYS concentrations 1, 10, and 100 mg L⁻¹, (I–K) BSA concentrations 1, 100, and 500 mg L⁻¹. Error bars are sample standard deviations from triplicate measurements.

4. Conclusions and implications

In this study, we systematically evaluated how CYS and BSA could change the stability, dissolution, and cell toxicity of AgNPs. Our results implied that both CYS and BSA can change the zeta potential and average diameter of AgNPs, this may influence the stability of AgNPs in water. UV-vis absorption spectroscopy method showed that both CYS and BSA could reduce the AgNPs concentration, but CYS may interact with AgNPs more effectively than BSA. Both CYS and BSA can weaken the toxicity of AgNPs. At higher concentrations of CYS, the toxicity of Ag⁺ and AgNPs was reduced dramatically. However cell viability decreased at higher BSA concentrations (≥50 mg L⁻¹). This may indicate that the transportation of AgNPs is more complex in the presence of CYS or BSA. Taken together, our study may offer meaningful and novel insights into the AgNPs potentially occurring in natural aquatic systems enriched with various types of DOM such as CYS and BSA.

Acknowledgements

This study was financially supported by the National Natural Science Foundation of China (51579099 and 51521006), the Program for Changjiang Scholars and Innovative Research Team in University (IRT-13R17), the Hunan Provincial Innovation Foundation for Postgraduate (CX2016B134).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra23675h

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