Serum albumin reduces the antibacterial and cytotoxic effects of hydrogel-embedded colloidal silver nanoparticles

Abstract

Although silver nanoparticles (AgNPs) are widely used as ion-releasing antimicrobial additives in medical devices, recent reports indicate the suppression of effectiveness in the presence of blood serum proteins. Bovine serum albumin (BSA) is known to bind silver and silver ions, so that the presence of proteins may change the antibacterial or cytotoxic properties of AgNPs even when they are embedded in a solid agar hydrogel matrix. We produced ligand-free AgNPs by laser ablation in water resulting in aqueous silver mass concentrations of 0.5 to 7.1%. The AgNPs were immersed into agar in concentrations of 5–70 μg ml⁻¹ medium. We examined the influence of 1% BSA within the hydrogel matrix on the nanoparticles’ antibacterial effect on four clinically relevant bacteria strains and the cytotoxicity of colloidal AgNP was tested on fibroblasts with or without 1% BSA. The hydrogel-immobilized AgNPs showed a significant reduction of antibacterial activity in the presence of BSA. Cytotoxicity started at a colloidal AgNP concentration of 35 μg ml⁻¹, and addition of BSA significantly reduced the effect on cell morphology and viability. Overall, in the presence of BSA, both antibacterial and cytotoxic effects of AgNPs were markedly reduced. Notably, a therapeutic AgNP window, requiring a dose at which pathogenic bacteria growth is inhibited while fibroblast viability is not affected, could only be observed in the absence of BSA. Addition of BSA reduces the antibacterial activity of AgNP to a point without significant growth inhibition of S. aureus but still observable cytotoxic effects on HGFib. Hence, the presence of a major blood serum protein significantly decreases the antimicrobial effects of AgNPs on a range of pathogenic bacteria even when the NPs are immobilized within an agar hydrogel model.

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Introduction

Silver ions are well known for their capacity to reduce bacterial growth.^1–4 Silver nanoparticles are used nowadays for their bactericidal properties in various applications such as filtration devices and textiles.^5,6 The coating of medical devices such as catheters, wound dressings or implants with AgNPs is also a growing market.^2,7–9 AgNPs are synthesized by various methods ranging from chemical precipitation to biological production by microorganisms,^10,11 where laser ablation in liquid is an alternative method to produce AgNPs of high purity without additional stabilizing agents or toxic byproducts.^12,13

Several studies have described the bactericidal effects of AgNPs in vitro on a variety of clinically relevant bacterial species.^1,14–16 It is generally accepted that AgNPs release Ag⁺ ions, which disrupt the bacterial cell membranes. The specific mechanism of this disruption process, however, is still a matter of extensive discussion.^17,18 Subsequently, silver ions also inhibit enzymatic activity within bacterial cells after their intrusion through the disrupted cell membrane.^19,20 The cytotoxic effects of AgNPs on eukaryotic cells and their potential danger for biological systems are also subjects of different studies.^21–25 It was demonstrated that the exposure of eukaryotic cells to AgNPs reduces the mitochondrial activity by inhibiting the mitochondrial respiratory chain, resulting in cellular degeneration.^26–28 It is likely that the biological activity of AgNPs on cells and bacteria depends on the size and morphology of the nanoparticles. Likewise, the measured effects are specific for different cell types and bacteria.^27,29

In a fluid medium proteins will adsorb onto the surface of nanoparticles.³⁰ The nature and the concentration of these proteins will not only determine the nanocolloid behavior, e.g. the stability against agglomeration, but may also affect cellular uptake, intracellular distribution and possible toxic effects.³¹ In particular, interactions with proteins may influence the surface chemistry of nanoparticles resulting in changes in their physicochemical parameters, such as their charge or agglomeration state, which directly affects their biological activity.^32–34 The role of these proteins is thus of major interest for the determination of antimicrobial and cytotoxic effects due to the binding of ion sources, e.g. AgNP-aggregation in media with a high electrolyte content.^35,36 In addition to the effect on nanoparticle dispersion, the presence of proteins may significantly reduce the bioactivity of silver in toxicity studies.³³ Since it is known that thiols bind to both heavy metal surfaces and ions, it is expected that proteins with amino acid sequences including the thiol-bearing cysteine may affect the dissolution and bioactivity of such nanoparticles. Accordingly, it recently has been shown that cysteine increases the release of the heavy metal ion nickel leached from colloidal nickel-containing nanoparticles. Surprisingly, the ion-release with the addition of increasing amounts of cysteine also reduced the bioactivity of the potentially toxic nickel colloid.³⁷ Overall, the literature indicates that proteins may affect ion release from the colloidal state and its resulting bioactivity, but the question arises if bioactivity reduction by BSA can also be observed if silver nanoparticles are immobilized in a solid matrix.

When embedded in a hydrogel-like agar, proteins are capable of diffusing within this matrix according to their size and the pore size of the hydrogel.³⁸ To study the release kinetics of proteins within hydrogels, bovine serum albumin (BSA) is a well established model protein.^39–41

Recently, it has been shown that AgNPs and silver ions significantly increase epithelial cell viability in combination with BSA, emphasizing that silver is bound to this protein.³³ Based on these investigations one may speculate that BSA also affects the antibacterial activity of silver nanoparticles even when embedded in a hydrogel-like agar matrix. To date, it is not known to what extent the binding of silver nanoparticles by BSA affects their antibacterial properties when embedded in hydrogels. Therefore, this study focuses on the influence of BSA on the antibacterial and cytotoxic effects of initially ligand-free colloidal AgNPs.

2. Materials & methods

2.1. Preparation of silver nanoparticles

Nanoparticles were generated by laser ablation of silver in deionized water under flow conditions. The silver target (Goodfellow, 99.99% purity) was irradiated using a Q-switched Nd:YAG slab laser (Edgewave HD40I-E) delivering 8 ns pulses at 3 kHz at a fundamental wavelength of 1064 nm and a pulse energy of 33 mJ. The laser beam was guided horizontally onto the target surface using a scanner system (Scanlab HurrySCAN II-14) with an f-theta lens of 56 mm focal length through a quartz glass window. The target was fixed in a self-constructed chamber and its surface was scanned in a spiral pattern during ablation. The liquid layer above the material surface was 5 mm. The nanoparticle concentration was measured gravimetrically by weighing the target before and after ablation. The process duration was 5 min and the resulting ablated mass was 93 mg with a colloidal silver nanoparticle concentration of 465 μg ml⁻¹. This colloid was diluted with deionized water for further characterization and biological testing. To ensure a uniform distribution of the nanoparticles within the liquid, each AgNP solution was ultrasonicated for 10 min before being used in any experiment.

2.2. Characterization of silver nanoparticles

The particle size distribution and the zeta potential were measured using dynamic light scattering (DLS) (Zetasizer ZS, Malvern Instruments Ltd.). All measurements were performed three times, and mean values and standard deviations were calculated. The absorption characteristics of colloidal AgNPs were characterized by UV/VIS spectrometry (Shimadzu 1650 PC). The morphology of the nanoparticles was characterized by dropping the colloidal solution onto a polished graphite target, followed by a subsequent drying and visualization by scanning electron microscopy (FEI Quanta 400).

2.3. Effects of AgNPs on bacteria

Four different bacterial strains Staphylococcus aureus (DSM 20231), Streptococcus salivarius (DSM 20067), Escherichia coli (DSM 1103) and Pseudomonas aeruginosa (ATCC BAA-47) were cultivated in Mueller-Hinton broth (CM0405, Oxoid) at 37 °C. Different quantities of dispersed AgNPs were added to Mueller-Hinton agar (1.5 wt%) which was premixed with BSA (1% w/v) resulting in final AgNP concentrations of 0.5 to 7.1 wt%, equal to a maximum dose of 5 to 70 μg Ag ions per ml liquid agar. For each strain and AgNP concentration, 4 plates with a diameter of 35 mm containing 2 ml of Mueller-Hinton agar were prepared. Also the same concentration range of AgNPs was added to Mueller-Hinton agar without BSA. Plates without nanoparticles were used as controls. After an overnight cultivation the bacteria were diluted 1 : 10⁶ to reach a final cell concentration of 10³ cells ml⁻¹ and 100 μl was plated onto each agar plate containing the immobilized AgNPs. After 24 h incubation at 37 °C, the number of colony forming units (CFU) on each plate was determined.

2.4. Cytotoxicity of AgNPs

Human gingival fibroblasts (HGFib, Cat. No.: 121 0412, Provitro) were cultivated and maintained in Dulbecco's Modified Eagle's Medium (DMEM) (FG0435, Biochrom) supplemented with 10% fetal bovine serum (FCS) (P270521, Pan-Biotech), 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin (A2212, Biochrom) at 37 °C in 5% CO₂ with 95% relative humidity.

HGFib cells were seeded at a density of 5 × 10³ cells per well in 96-well plates for the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The test for mitochondrial function of HGFib cells was performed by a MTT viability assay according to the protocol of the manufacturer (Cell Proliferation Kit 1, Roche Diagnostics). In living cells, the dye MTT was used to determine the mitochondrial activity of living cells when under the cytotoxic influence of AgNPs. Therefore the reduction to purple formazan by enzymatic reaction was used as an indicator for the viability of the HGFib cells. After 24 h of cultivation, the growth medium was removed and 100 μl fresh DMEM FCS medium including different amounts of AgNPs were added. In agreement with our observations, Mahl et al. recently showed that AgNP aggregation in cell culture media is prevented by addition of FCS to the medium where the nanoparticles remained well dispersed above 1 wt% protein concentration.³¹

For each concentration of AgNPs (5, 10, 20, 35 and 70 μg ml⁻¹), four wells were incubated with or without 1% w/v BSA at 37 °C. After 24 h of incubation, 10 μl of staining solution was added to each well. Following 4 h of further incubation, 100 μl of the solubilization solution were added to each well and the plates were incubated overnight at 37 °C. Afterwards, the absorbance of the formed formazan was measured at a wavelength of 540 nm – with 650 nm as reference – using a fluorescence microplate reader (Infinite F200, Tecan).

2.5. CLSM characterization of cytotoxic effects

HGFib cells were seeded at a density of 5 × 10³ cells per well in 96-well plates for confocal laser scanning microscopy (CLSM). After 24 h of stimulation under the respective experimental settings (see section 2.4), the cells were washed with DMEM. The fluorescent dye calcein (Invitrogen) was added to a final concentration of 8 μg ml⁻¹ for 5 min and the cells were finally washed with DMEM. The morphology of the cells was documented with a Leica inverse confocal laser scanning microscope (Leica DM IRB, Leica) by a 20× dry objective with 0.3 numerical aperture.

2.6 Statistical analysis

Experiments on antibacterial activity were performed four times; experiments on cytotoxicity were done in triplicate. All results are presented as mean ± standard deviation. Group differences were analyzed by a Student's t-test. Statistical significance was accepted at a level of P ≤ 0.05.

Results

3.1. Properties of silver nanoparticles generated by pulsed laser ablation

The hydrodynamic particle size distribution is displayed in Fig. 1a and ranges from 10 to 60 nm following a lognormal-function, which is characteristic for laser-generated particles.⁴² The peak of the function at 21 nm specifies the average hydrodynamic particle diameter. It has to be noted that, in contrast to the feret diameter (metal core), the hydrodynamic diameter includes the hydrate shell surrounding the nanoparticles. The most frequent particle shape is spherical, as is always the case during pulsed laser ablation in ligand-free liquid,⁴³ in contrast to pulsed laser ablation with intended structure dirigism by post irradiation.⁴⁴

Fig. 1 a) Hydrodynamic size distribution of laser-generated silver nanoparticles (fitted with a lognormal-function) by dynamic light scattering (DLS); b) UV/VIS absorption spectrum and c) scanning electron micrograph of silver nanoparticles that were embedded into agar resulting in a solid nanoparticle composite; the resolution of the SEM was 2nm.

Fig. 1b shows the absorption characteristics of the silver nanoparticle colloids and the characteristic plasmonic resonance peak of silver nanoparticles at 410 nm. This peak reveals the presence of stable nanoparticles in the diluted sample. Accordingly, the zeta potential of the colloidal silver nanoparticles is −24 mV ± 1 mV, indicating stable nanoparticles. The scanning electron micrograph in Fig. 1c shows overall spherical laser-generated nanoparticles.

3.2 Bactericidal effects

AgNPs showed different degrees of antibacterial effects within the applied concentration range of 5–70 μg ml⁻¹ depending on the bacterial species treated. P. aeruginosa was most sensitive to AgNPs, demonstrating a complete inhibition of the bacterial growth at a concentration of 10 μg ml⁻¹ (Fig. 2d). The number of CFU of the gram-negative species E. coli was inhibited to 70% of the control treatment at the same concentration of AgNPs. We observed no growth of E. coli at a concentration of 20 μg ml⁻¹ (Fig. 2c). A complete growth inhibition of the gram-positive bacteria S. salivarius and S. aureus was achieved in the presence of 35 μg ml⁻¹ and 70 μg ml⁻¹ AgNPs, respectively (Fig. 2a and 2b).

Fig. 2 Bactericidal effects of laser-generated AgNPs on a) S. aureus, b) S. salivarius, c) E. coli, d) P. aeruginosa after in vitro cultivation on Mueller-Hinton agar plates with or without BSA (1% w/v) over 24 h. * = Statistically significant difference between groups (P ≤ 0.05).

In comparison, the addition of BSA led to an increase of the effective antibacterial concentration of AgNP. All four bacterial species showed a complete inhibition of growth at a twofold or higher AgNP concentration in the presence of BSA when compared to the experiments without BSA. P. aeruginosa and E. coli were completely inhibited at an AgNP-concentration of 35 μg ml⁻¹ in the presence of BSA. The growth of S. salivarius was completely eliminated at a concentration of 70 μg ml⁻¹, whereas the growth of S. aureus could not be inhibited completely by any of the applied AgNP-concentrations in the presence of BSA.

3.3 Cytotoxic effects

The result of the biocompatibility assay demonstrated that mitochondrial activity, visualized by MTT conversion, was reduced with increasing concentrations of AgNPs. In contrast to a moderate inhibition of mitochondrial activity by low concentrations of AgNPs, it was completely reduced by a concentration of 35 μg ml⁻¹ or higher.

In the presence of BSA a minimal reduction of mitochondrial activity was also observed for low concentrations but inhibition was only moderate at a concentration of 70 μg ml⁻¹ in the presence of BSA (Fig. 3).

Fig. 3 The cytotoxic effects of colloidal AgNPs on HGFib with or without added BSA (1% w/v) by MTT assays. * = Statistically significant difference between groups (P ≤ 0.05).

3.4 Cell morphology

Without the addition of BSA, calcein-stained HGFib showed the expected spindle-shaped cell morphology and revealed no obvious signs of growth inhibition in the presence of AgNP concentrations of 5 to 10 μg ml⁻¹ (Fig. 4 I–III). A concentration of 20 μg ml⁻¹ AgNPs led to a decreased number of adhering HGFib in some areas (Fig. 4 IV) and at 35 μg ml⁻¹ the cells were round, less attached and less dense. Due to the decreased attachment, HGFib became more susceptible to necessary washing procedures in the staining protocol and therefore was washed off completely at a concentration of 70 μg ml⁻¹ (Fig. 4 V–VI).

Fig. 4 Calcein stained human gingival fibroblasts treated with different concentrations of silver nanoparticles; I) control, II) 5 μg ml⁻¹, III) 10 μg ml⁻¹, IV) 20 μg ml⁻¹, V) 35 μg ml⁻¹, VI) 70 μg ml⁻¹; (a) without BSA, (b) with 1% (w/v) BSA. Pictures obtained by CLSM (20× objective), scale bar = 200 μm

The addition of BSA significantly reduced the cytotoxic effects of AgNPs on fibroblasts at concentrations between 10 and 70 μg ml⁻¹ of AgNPs (Fig. 3). In the presence of BSA under otherwise identical conditions a complete cell detachment was not observed even at a concentration of 70 μg ml⁻¹ AgNPs.

Discussion

AgNPs were produced by laser ablation in liquid, which has the biologically-relevant advantage of the synthesis of clean nanoparticles.^10,12,45,46 Laser ablation was performed in deionized water under controlled beam settings, resulting in particles with ligand-free surfaces. However, the relatively high absorption of the dispersion at higher wavelengths indicates the presence of agglomerates. This is probably due to the high concentration of the stock solution which increases the probability of particle–particle interactions. The ultrasonication and dilution of the AgNPs before each experiment provided an evenly distributed colloid. The average particle diameter of 21 nm was comparable with many studies reporting about the antibacterial and cytotoxic effects of AgNPs.^{1,15,20,24,32,34,47–50}

The colloidal nanoparticles were embedded into Muller-Hinton agar medium, mimicking hydrogel–nanoparticle-composites used in biomedical applications. Agar was used as a hydrogel model in order to allow comparability with standard cell culture tests. In addition, agar has a high water uptake ensuring a high ion release.⁵¹ Regarding the antimicrobial effects of agar-immobilized AgNPs, the tested bacterial species showed different growth behaviors when cultivated with or without the addition of 1% w/v BSA. Without BSA, strong antimicrobial effects of the AgNPs were observed at a concentration range from 10 to 70 μg ml⁻¹. This observed antibacterial effect from immobilized AgNPs without BSA indicates a release of Ag⁺-ions from agar as it is known from AgNPs embedded in polymer composites.⁵² The gram-negative bacteria E. coli and P. aeruginosa were more sensitive to immobilized AgNPs than the gram-positive bacteria S. aureus and S. salivarius. These results are in agreement with a study by Ghosh et al., who discovered a similar inhibition of E. coli and S. aureus within the same concentration range of agar-immobilized AgNPs with a particle diameter ranging from 15 to 25 nm.⁴⁷ Morones et al. reported similar antimicrobial activities for E. coli and P. aeruginosa using a comparable particle diameter of 21 nm.¹⁵ Both groups used hydrogels with suspended nanoparticles which were chemically synthesized. Despite the different methods of synthesis the result was comparable showing the comparability of laser-generated AgNPs. Different antibacterial effects of AgNPs on gram-positive or gram-negative bacteria were also reported by other groups and are likely caused by the differences between the chemical compositions of their membranes.^{1,11,34,47,53} Other groups reported a complete inhibition of bacterial growth at even lower concentrations of AgNPs.^1,17,19,48 These discrepancies are believed to result from the different sizes and surface characteristics of the silver nanoparticles used. Although these studies revealed that the size and even the shape of nanoparticles are the main factors influencing their antimicrobial effects,^15,17,19,20 the most important one for their bactericidal effect is the release of Ag⁺-ions by the nanocolloids in proximity to the bacterial cell membrane^1,14,15,20 which is difficult to quantify in hydrogel agar. Thus, although the influence of particle size and shape has an effect on bacteria as mentioned before, the aim of this study was to determine the influence of serum albumin.

The role of proteins in general and BSA in particular on the antibacterial effect of silver nanoparticles is only rarely investigated in contrast to their effects on biocompatibility. The reduction of the antimicrobial effect of AgNPs by BSA was significant for all bacterial strains tested in this study. The toxic effect of Ag⁺ is known to be reduced when Ag⁺-ions form complexes with thiols or counter-anions present in solution.⁵⁴ Since the presence of BSA in agar reduces the antibacterial effect and BSA was reported to diffuse in agar hydrogel,⁴⁰ it can reasonably be concluded that BSA binds to the silver particles causing a reduction of their antimicrobial effectiveness. This hypothesis is supported by the results of two recent studies in which BSA and cysteine reduced the bioactivity of ion-releasing nanoparticles, even though ion dissolution was increased by cysteine.^33,37 Accordingly, in our study the antimicrobial effectiveness of the AgNPs was significantly reduced for all investigated strains.

Since reduction of bioactivity is expected to affect the dose thresholds of the therapeutic window, the effective threshold and the toxic threshold, we tested the effect of BSA on the biocompatibility of AgNPs as well. HGFib was co-incubated with colloidal AgNPs in a growth medium with or without the addition of 1% w/v BSA. A MTT assay was used to determine the viability of HGFib through the reduction of MTT dye. Dark blue formazan is formed by the activity of the enzyme succinate dehydrogenase, which is present in cell mitochondria. Without BSA, the cytotoxicity assay demonstrated a reduced MTT conversion, which indicates a significant inhibition of mitochondrial activity in the cells at an AgNP concentration of 35 μg ml⁻¹. There was only a limited decline of mitochondrial activity up to a concentration of 20 μg ml⁻¹ AgNPs. Therefore, it can be assumed that a concentration of 35 μg ml⁻¹ is a threshold for laser-generated AgNPs, whereas higher concentrations show a strong cytotoxic effect on HGFib.

After the addition of BSA, the cytotoxic effects of AgNPs were significantly reduced. Since the AgNPs are in the immediate proximity of the cells, the binding of BSA molecules to the AgNPs and the following coverage of their surfaces as well as the binding or complexing of the silver ions are key factors for their activity loss.^{55,17,30,56,57}

The results of the MTT assay are supported by the CLSM images of the HGFib cells which were cultivated with the same concentration of AgNPs with or without BSA. Without BSA, an increasing number of detached cells were formed, starting at an AgNP concentration of 20 μg ml⁻¹, which indicates a cytotoxic effect on the cells. By adding BSA to the medium, the detachment of cells was reduced and rounded cells were only found at higher concentrations of AgNPs. These observations are in accordance with other studies that report the enhancement of the biocompatibility of silver nanoparticles by an addition of BSA. The effects were also described by Kittler et al. for human mesenchymal stem cells which were treated with polyvinylpyrrolidone-coated silver nanoparticles. They reported that the addition of BSA led to the binding of released silver ions.³³ Additionally, the effects of fetal calf serum (FCS) on the cytotoxicity of AgNPs should be mentioned. Kittler et al. reported a strong decrease of the cytotoxic effect of AgNPs when they were suspended in growth medium containing FCS.³³ However, many components of cultivation media may affect the surface properties of nanoparticles and might therefore change their bioactivity, although such a detailed analysis of the components of the HGFib cultivation medium and its effect on the AgNPs was not the aim of the present study and needs further investigations.^34,58

The threshold dose values for the cytotoxic effect of AgNPs are consistent with other studies examining the biocompatibility of these particles, considering that in general these effects were noticed at highly variable concentration ranges for diverse cell lines.^{23,33,47,48,50,59}

Conclusion

Although albumin is the dominant protein in blood serum and is known to be able to diffuse in solid hydrogels, its effect on the bioactivity of ion releasing nanoparticles and nanomaterials is often underestimated during in vitro bio-response studies. Notably, bovine serum albumin significantly reduces the antimicrobial effectiveness of silver nanoparticle hydrogel–composites. BSA decreases the antibacterial effect on Staphylococcus aureus, Streptococcus salivarius, Escherichia coli and Pseudomonas aeruginosa on solid growth media, when both nanoparticles and BSA are embedded in an agar matrix. In addition, we observed a decreased cytotoxicity of colloidal AgNPs against human gingival fibroblasts in the presence of BSA. The reduction of both the effectiveness (against bacteria) and the toxicity (against fibroblasts) caused by the presence of BSA significantly narrowed the therapeutic window, and in the case of Staphylococcus aureus fully diminished the therapeutic applicability of silver nanoparticles.

The solid agar matrix used to embed the nanoparticles in our investigation represents the standard material in microbiological cultivation, contributing to understand the strong influence of serum proteins on the bio-response and applicability of silver nanoparticles and silver nanoparticle composites.

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Footnote

† Equal contributors.

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