Article pubs.acs.org/Langmuir
Cytotoxicity Induced by Engineered Silver Nanocrystallites Is Dependent on Surface Coatings and Cell Types Anil K. Suresh,*,†,∥ Dale A. Pelletier,† Wei Wang,‡ Jennifer L. Morrell-Falvey,† Baohua Gu,‡ and Mitchel J. Doktycz*,†,§ †
Biosciences Division, ‡Environmental Sciences Division, and §Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, Tennessee, 37831 United States S Supporting Information *
ABSTRACT: Due to their unique antimicrobial properties silver nanocrystallites have garnered substantial attention and are used extensively for biomedical applications as an additive to wound dressings, surgical instruments and bone substitute materials. They are also released into unintended locations such as the environment or biosphere. Therefore it is imperative to understand the potential interactions, fate and transport of nanoparticles with environmental biotic systems. Numerous factors including the composition, size, shape, surface charge, and capping molecule of nanoparticles are known to influence cell cytotoxicity. Our results demonstrate that the physical/ chemical properties of the silver nanoparticles including surface charge, differential binding and aggregation potential, which are influenced by the surface coatings, are a major determining factor in eliciting cytotoxicity and in dictating potential cellular interactions. In the present investigation, silver nanocrystallites with nearly uniform size and shape distribution but with different surface coatings, imparting overall high negativity to high positivity, were synthesized. These nanoparticles included poly(diallyldimethylammonium) chloride-Ag, biogenic-Ag, colloidal-Ag (uncoated), and oleate-Ag with zeta potentials +45 ± 5, −12 ± 2, −42 ± 5, and −45 ± 5 mV, respectively; the particles were purified and thoroughly characterized so as to avoid false cytotoxicity interpretations. A systematic investigation on the cytotoxic effects, cellular response, and membrane damage caused by these four different silver nanoparticles was carried out using multiple toxicity measurements on mouse macrophage (RAW-264.7) and lung epithelial (C-10) cell lines. Our results clearly indicate that the cytotoxicity was dependent on various factors such as surface charge and coating materials used in the synthesis, particle aggregation, and the cell-type for the different silver nanoparticles that were investigated. Poly(diallyldimethylammonium)-coated Ag nanoparticles were found to be the most toxic, followed by biogenic-Ag and oleate-Ag nanoparticles, whereas uncoated or colloidal silver nanoparticles were found to be the least toxic to both macrophage and lung epithelial cells. Also, based on our cytotoxicity interpretations, lung epithelial cells were found to be more resistant to the silver nanoparticles than the macrophage cells, regardless of the surface coating.
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INTRODUCTION Interest in nanomaterials with controlled structures and functionality is growing rapidly. Nanomaterials are being produced in large quantities for implementation in various consumer products such as creams and ointments, cosmetics, paint and fuel additives, and solar energy applications.1,2 Due to their large surface area, high catalytic activity, and intrinsic physicochemical, optoelectronic, and biological properties, nanomaterials are also finding applications in biosensors3 as well as medical and electronic devices.4 Silver nanocrystallites are of particular interest because of their well-known bactericidal and fungicidal properties and are widely used in medical devices, textiles, food packaging, and healthcare and household products.1,3,5,6 One type of silver nanomaterial, silver sulfadiazine, is used clinically to reduce burn or wound infections caused by multidrug resistant bacteria and fungi.7,8 Silver nanoparticles are also used in water purification and air quality management.1 Due to widespread use and exposure to © 2012 American Chemical Society
these nanomaterials, there is ever-growing concern regarding their potential detrimental impacts on the environment. The same properties that make silver nanoparticles useful in various applications however may have adverse effects on environmental systems. To this end, a number of studies have addressed the potential toxicity of silver nanocrystallites on different cell systems including bacteria, fungi, and mammalian cells.7−12 From such studies, the cytotoxicity of silver nanoparticles has been attributed to several possible mechanisms, including the dissolution or release of Ag ions from the nanoparticles,13 disruption of cell-membrane integrity,10 oxidative stress,14 protein or DNA binding and damage,1 reactive oxygen species generation,15 and apoptotic cell death.16 It seems likely that the Received: May 12, 2011 Revised: December 15, 2011 Published: January 4, 2012 2727
dx.doi.org/10.1021/la2042058 | Langmuir 2012, 28, 2727−2735
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atmosphere. For each treatment the cells were washed twice with PBS and then incubated with a range of concentrations of different silver nanoparticles for 4 and 12 h. As the cytotoxicity trends for both these time points were similar, data for only 4 h is shown. Cells treated with the different capping agents alone and cells treated with silver nitrate (AgNO3), served as controls. For statistical data analysis and to ensure reproducibility, each treatment was performed in octuplet and each assay was repeated at least three times. After exposure to the nanoparticles, the cells were rinsed once with PBS, 200 μL of supplemented RPMI was added and the plate was incubated at 37 °C for 18−24 h. The MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide) was then added to each well (250 μg/ mL) and the cells were reincubated at 37 °C for additional 4 h. After a final wash with PBS, the PBS was aspirated and 200 μL of DMSO was added to each well and the absorbance at 560 nm was measured using a spectrophotometer. Membrane Integrity Assessment by Lactate Dehydrogenase Assay. Membrane integrity following treatment of cells with silver nanoparticles was evaluated by measuring lactate dehydrogenase (LDH) enzyme activity in cell supernatant using the TOX-7 kit (Sigma Aldrich) as instructed by the manufacturer. Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) measurements. To determine the dissolution of silver ions for the four types of silver nanoparticles, ICP-MS measurements (PerkinElmer, Shelton) were performed as described earlier.10 Briefly, 5 mL each of the various nanoparticle suspensions was placed into dialysis tubing (3500 Da) and dialyzed against 20 mL of water or RPMI for ∼80 h. After which, the dissolved Ag in the external suspension was measured by ICP-MS. Qualitative Observation of Cell Morphology by Phase Contrast Microscopy. Cells were cultured as described for the MTT assay. For these experiments, cells were passaged onto 50 mm glass bottomed dishes (MatTek Corporation, Ashland, MA) at a density of 2 × 10 6 cells, in 3 mL supplemented RPMI. After 2 days, the growth medium was removed by aspiration and fresh, low nutrient OPTIMEM medium (Invitrogen) containing silver nanoparticles was added. Various concentrations of nanoparticles were tested, representing approximately IC70−80 based on data from the MTT assay. The cells were incubated for an additional 1 h, washed three times with PBS, and observed by microscopy. Membrane Integrity as Determined by LIVE/DEAD Staining and Confocal Microscopy. Cells were cultured and treated with various silver nanoparticles as described above. Following nanoparticle exposure, the cells were stained with propidium iodide and Syto9 (Invitrogen) as per the manufacturer’s protocol and the membrane integrity was assessed using confocal microscopy imaging.
mechanism of toxicity depends on properties of the nanoparticles, such as the surface area, size and shape, capping agent, surface charge, purity of the particles, structural distortion and bioavailability of the individual particles.10 Despite efforts by several investigators, the exact mechanisms and material dependence of nanoparticle induced toxicity remains unclear. In the present study, we investigate for the first time comparative analyses on the effects of various surface coatings and the methodology employed to fabricate different silver nanocrystallites on two different cell lines, mouse macrophage and lung epithelial cells. Relative cytotoxicity was based on 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Additionally, Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) measurements and fluorescent staining followed by microscopy were used to further assess cytotoxicity mechanism and potential membrane damage. Different chemical and or biological coatings on the nanoparticle surface clearly contribute to the toxic effects of nanoparticles and can potentially guide the use of nanoparticles for intended applications and for understanding the potential consequences of unintended exposures.
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EXPERIMENTAL SECTION
Mouse macrophage (RAW 264.7)17 and lung epithelial (C-10)18 cell lines were used in our studies. All chemicals and reagents were from standard commercial sources and of the highest quality available. Synthesis and Purification of Silver Nanocrystallites. Different silver nanocrystallites usedpoly(diallyldimethylammonium)-Ag (PDADMAC-Ag),19 biogenic-Ag,10 uncoated-Ag, and oleate-Ag20,21 were synthesized and thoroughly characterized as described earlier. The synthesized particles were purified by filtering the nanoparticle suspension through a sterile 0.1 μm syringe filter, followed by ultracentrifugation (100000g, 1 h). After washing twice with Milli Q water the nanoparticles were used for cytotoxicity experiments. To determine the nanoparticle concentration, a 50 mL aliquot of each of the purified nanoparticle suspensions was dried followed by weighing and quantifying the mass of silver nanoparticles. Physical Characterization. UV−vis absorbance spectra were recorded on a CARY 100 Bio spectrophotometer (Varian Instruments, CA) operated at a resolution of 1 nm. Fluorescence measurements were performed on a BioTek Synergy 2 micro plate reader (BioTek Instruments Inc., Vermont, PN) in black 96-well plates. Dynamic light scattering and zeta potential measurements were performed on a Brookhaven 90 Plus/BI-MAS Instrument (Brookhaven Instruments, New York). Fourier transform infrared (FTIR) spectroscopy analysis of the silver nanoparticles deposited on a ZnSe window were carried out on a Nicolet Magna-IR 760 spectrophotometer at 4 cm−1 resolution. Transmission electron microscopy (TEM) measurements for the samples prepared on carbon-coated copper grids were performed on a Hitachi HD-2000 STEM operated at an accelerating voltage of 200 kV. Phase contrast microscopy imaging was performed on an Olympus IX70 inverted microscope, equipped with a Nuance multispectral imaging system (including a visible liquid-crystal filter tunable between 420 and 750 nm). Confocal microscopy images were obtained using a Zeiss LSM 710 confocal laser-scanning microscope with a Plan-Apochromat 20x/0.8 objective (Carl Zeiss Microimaging, Thornwood, NY). Optical sections were collected at 1 μm spacing and shown as a maximum intensity projection using Zen 2009 software (Carl Zeiss). Membrane Function by MTT Assay. Cytotoxicity assessment was performed using the MTT assay as described earlier with slight modifications.22 Briefly, the cells were seeded at 5 × 103 per well in a 96-well tissue culture plate and grown to 80% confluence in 200 μL of Roswell Park Memorial Institute medium (RPMI) (Invitrogen) supplemented with 0.2 mM L-glutamine, 100 U mL−1 penicillin, 100 μg mL−1 streptomycin and 10% FBS in a 5% CO2 humidified
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RESULTS AND DISCUSSION Due to their unique antimicrobial properties silver nanocrystallites are implemented in various medical applications for the treatment of infections on wounds and burns caused by multidrug resistant bacteria and fungi. The toxicity of silver nanoparticles is well established with respect to prokaryotic,10 eukaryotic,5 as well as aquatic cell systems23 and raises concerns about adverse impacts due to unintentional exposures. The toxicity of silver nanocrystallites can be correlated to their size and shape, surface charge, capping agent, remnants of solvents and surfactants used in their synthesis and other properties. To have a better understanding on the contribution of these factors, the cytotoxicity of four different types of surface engineered silver nanoparticles including PDADMAC-Ag containing a poly(diallyldimethylammonium) surface coat,19 biogenic-Ag containing a protein or peptide surface coat,10 and oleate-Ag with oleate surface coat20 were compared to uncoated-Ag, which likely contains adsorbed nitrate on their surface,21 and assessed using mouse macrophage and lung epithelial cells. The detailed synthesis methodologies, as well as characterizations in terms of purity, morphology, crystallinity 2728
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Figure 1. TEM and FTIR analyses of the four different types of silver nanoparticles used in the cytotoxicity studies. TEM images of (A) PDADMAC-Ag, (B) biogenic-Ag, (C) uncoated-Ag, and (D) oleate-Ag nanoparticles; (E−H) plots of their respective particle histogram size distributions. Fourier transform infrared spectrum of (I) PDADMAC-Ag, (J) biogenic-Ag, (K) uncoated-Ag, and (L) oleate-Ag nanoparticles. Significant vibration bands are labeled.
Table 1. Surface Charge, Size Distributions and Intermediate Cytotoxicity Concentrations (IC50) of the Four Differently Surface Coated Silver Nanoparticles on Mouse Macrophage and Lung Epithelial Cells inhibitory concentration (μg/mL) silver nanoparticle type PDADMAC-Ag Biogenic-Ag Uncoated-Ag Oleate-Ag
mean diameter (nm) based on TEM ∼4.5 ∼4.0 ∼9.0 ∼4.0
± ± ± ±
1.5 1.5 2 1
sizes based on DLS (nm) in water
sizes based on DLS (nm) in (RPMI)
∼43.4 ∼82.5 ∼62.6 ∼46.3
∼164 ∼103.6 ∼137.8 ∼77.2
zeta potential (mV) +45 −12 −42.5 −45.8
± ± ± ±
3.1 2 5.2 4.4
macrophage cells
lung epithelial cells
∼0.1 ∼0.125 ∼4.9 ∼1.1
∼0.45 ∼0.7 ∼6.3 ∼1.6
H). Previous reports suggested that size and shape might play a role in determining bactericidal activity24 as well as the eukaryotic cell cytotoxicity25 of silver nanoparticles. A decrease in cell cytotoxicity has been correlated with an increase in particle size.25 The contribution of size to cell cytotoxicity is minimized in our study since all four nanoparticles were of similar size and shape distributions (Figure 1). Thus other basic surface properties that may contribute to the overall cytotoxicity including the hydrodynamic sizes, surface coatings and the surface charges of the particles were examined (see Table 1 and Figure 1I−L). The hydrodynamic diameter of the particles analyzed by dynamic light scattering (DLS) measurements, both in Milli Q water and RPMI used for the cytotoxicity experiments, appeared to be larger than the diameters determined by TEM (Figure 1). This discrepancy may be attributed to particle aggregation in the DLS measurements while TEM allows latitude for eliminating clumps of particles.10 Larger aggregates were observed in
and surface properties of the silver nanoparticles have been described previously.10,20,21 To draw accurate conclusions the particles were extensively characterized as particle characteristics are known to contribute to cytotoxicity. Moreover, the use of toxic solvents, surfactants and precursors were avoided to prevent false toxicity interpretations. In addition, the particles were cleaned extensively prior to experimentation to avoid the interference of impurities and remnants in toxicity assessments. Irrespective of the synthesis methodology employed, all four types of silver nanoparticles examined were similar in size (
