Effect of Protein Corona on Silver Nanoparticle Stabilization and Ion

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Effect of protein corona on silver nanoparticle stabilization and ion release kinetics in artificial seawater Maja Levak, Petra Buri#, Maja Dutour Sikiri#, Darija Domazet Jurašin, Nevenka Mikac, Niko Ba#i#, Roland Drexel, Florian Meier, Željko Jakši#, and Daniel Mark Lyons Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03161 • Publication Date (Web): 11 Jan 2017 Downloaded from http://pubs.acs.org on January 13, 2017

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Environmental Science & Technology es-2016-03161y

Effect of protein corona on silver nanoparticle stabilization and ion release kinetics in artificial seawater

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Revised

Maja Levak†, Petra Burić†, Maja Dutour Sikirić††, Darija Domazet Jurašin††, Nevenka Mikac‡, Niko Bačić‡, Roland Drexel§, Florian Meier§, Željko Jakšić †, Daniel M. Lyons†* † †† ‡

§

*

Ruđer Bošković Institute, Center for Marine Research, G. Paliaga 5, 52210 Rovinj, Croatia Ruđer Bošković Institute, Division of Physical Chemistry, Bijenička cesta 54, 10000 Zagreb, Croatia Ruđer Bošković Institute, Division of Marine and Environmental Research, Bijenička cesta 54, 10000 Zagreb, Croatia Postnova Analytics GmbH, Max-Planck-Straße 14, 86899 Landsberg am Lech, Germany

To whom correspondence may be addressed. Tel. +385 52 804725; Fax +385 52 804780; E-mail: [email protected]

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Abstract

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In parallel with the growing use of nanoparticle-containing products, their release into the

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environment over the coming years is expected to increase significantly. With many large

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population centers located in near-coastal areas, and increasing evidence that various

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nanoparticles may be toxic to a range of organisms, biota in estuarine and coastal waters may

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be particularly vulnerable. While size effects may be important in cases, silver nanoparticles

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have been found to be toxic in large part due to their release of silver ions. However, there is

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relatively little data available on how nanoparticle coatings can affect silver ion release in

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estuarine or marine waters. We have found that albumin, as a model for bio-corona -forming

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macromolecules which nanoparticles may encounter in wastewater streams, stabilizes silver

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colloids from agglomeration in high salinity marine waters by electrosteric repulsion for long

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time periods. A minimum mass ratio of about 130 for albumin:silver nanoparticles (40 nm)

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was required for stable dispersion in seawater. Increasing albumin concentration was also

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found to reduce dissolution of nanoparticles in seawater with up to 3.3 times lower

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concentrations of silver ions noted. Persistent colloids and slow sustained ion release may

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have important consequences for biota in these environmental compartments.

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ACS Paragon Plus Environment


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Introduction

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Over the past decade there has been a rapidly growing number of industrial

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applications and consumer products that contain engineered nanoparticles (ENPs).1-4 With

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this increasing use of ENPs there is growing concern that they may reach the environment in

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sufficient quantities to give rise to significant deleterious effects on living organisms.5-7

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During use or at the end of product life cycle ENPs may be released incidentally into the

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environment, for example via household waste water streams, from industry by direct

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outflow, atmospheric deposition, materials degradation through weathering, rainwater runoff

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and from wastewater treatment plants.8 Much of the research on the behavior and fate of

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ENPs in the environment has focused on terrestrial aquatic systems (lakes, rivers and

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groundwater) in part due to the expectation that ENPs would remain sufficiently dispersed in

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freshwater systems as to pose the greatest exposure threat to biota living in those

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environmental compartments. The relative stability of nanoparticles in freshwater derives

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from ENP stabilization by natural organic matter and the moderately low ionic strength of the

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media. Over time ENPs may be transported further and, for example if not already

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transformed by redox processes, would gradually reach estuarine waters where rapid

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agglomeration and precipitation would ensue due to the increasing salt content. However,

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there is increasing evidence that individual nanoparticles or small agglomerates can persist for

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up to periods of days in high salt content media such as estuarine and marine waters, likely

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through stabilization by humic substances9, polysaccharides10 or adsorption on organic

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detritus.11 As more than half the world’s population lives in near-coastal areas, it is

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increasingly likely that significant quantities of ENPs will reach coastal marine waters before

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degradation processes can sufficiently reduce their concentration. Indeed, it has been recently

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shown that extremely high concentrations of ENPs such as titanium dioxide can be present in

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coastal waters just as a result of sun protection creams alone.12 Somewhat belatedly, research

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Revised

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is only now beginning to address the behavior of ENPs entering estuaries and coastal waters

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and their fate is still not fully understood. The importance of this is further highlighted by the

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fact that commercially-important marine organisms have shown the ability to accumulate

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ENPs in various ways, by either filtering water,13 ingestion,14 deposit feeding 15 or by trophic

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transfer.16 Indeed, bioaccumulation and trophic transfer have the potential to transport ENPs

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up the food chain and eventually impact on humans. Further, ENPs have been shown to cause

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a range of adverse effects in aquatic organisms including disruption of cell membranes (which

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impacts on nutrient transport into the cells), extensive generation of reactive oxygen species

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(ROS), deterioration of DNA integrity and degradation of proteins.17

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Currently one of the most broadly used ENPs are silver nanoparticles (AgNPs) due to

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their electrical and optical properties, and the textile, food and cosmetic industries employ

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them in a wide range of products due to their antimicrobial abilities.18 AgNPs are typically

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stabilized by coating with various capping agents to provide electrostatic, steric or

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electrosteric repulsion, with citrate, oleic or tannic acid, gum arabic, polyvinylpyrrolidone

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(PVP) and polyethylene glycol (PEG) commonly being used.19 While the electrical double

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layer acts to maintain their electrostatic equilibrium and minimize surface energy, the

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thickness of this electric double layer is reduced, and hence colloid stability decreases, as the

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ionic strength of the media increases.20 Thus, considering that ionic strength in freshwater is

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about 1-10 mM, and in seawater is about 700 mM,19 it is likely that such coated AgNPs in

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brackish and marine waters will form large aggregates. Indeed, the salt-induced collapse of

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the electric double layer and bridging effects which can occur if capping agents form

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complexes with divalent cations 21 lead to eventual aggregation of the nanoparticles alone

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(homoaggregation)

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(heteroaggregation).5 However, it has been found that this process is concentration-dependent

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where ENPs at very low concentrations have little or no possibility for physical contact

or

aggregation

with

natural

colloids

3


in

the

water

column



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among themselves,13 resulting in the likelihood that they remain as individual nanoparticles

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stabilized by natural organic matter such as phytoplankton exudates.10 To date there are

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relatively few studies on the physico-chemical behavior of ENPs in seawater,22-28 and data on

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their stability is equivocal with reports showing either rapid agglomeration or significant

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persistence of nanoparticles. Further, these nanoparticles were typically coated with ions such

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as citrate or with PEG, while little attention has been paid to the possibility of biomolecules

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present in the environment displacing these ions on AgNPs. Indeed, as AgNPs move through

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various environmental compartments, it is likely that they will acquire a bio-corona due to

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interactions with, for example, microbes and their exudates which will impact not only on

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AgNPs stability but also on the kinetics of silver ion (Ag+) release from the nanoparticles. The

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latter is an important point as much current research indicates that Ag+ ions present greater

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toxic potential to biota than AgNPs.19 Thus, in order to mitigate potentially adverse effects to

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biota in brackish and coastal waters the physico-chemical behavior of ENPs in these aquatic

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environmental compartments must be thoroughly resolved as a first step towards developing

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predictive models. In this direction we investigated the stability, agglomeration state,

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persistence and ion release kinetics of protein-stabilized AgNPs in artificial seawater (ASW)

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solutions as a model for biomolecule-coated AgNPs in brackish and coastal waters.

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Experimental

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Citrate-stabilized silver nanoparticle (AgNP) stock suspensions (20 µg mL-1) of 40

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nm, bovine serum albumin (BSA), NaOH, CaCl2 and MgSO4·7H2O were purchased from

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Sigma-Aldrich (St. Louis, MO, USA). NaCl, KCl, and MgCl2·6H2O were purchased from

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Kemika (Zagreb, Croatia). All chemicals were of high analytical grade and used as received.

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Ultrapure water (18.2 MΩ) was provided by a Millipore Advantage System (Merck Millipore,

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Darmstadt, Germany).

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Revised

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Concentrated artificial seawater (ASW; salinity (S) 108.7) was prepared by dissolving

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NaCl (450 mmol), KCl (9.93 mmol), CaCl2 (8.92 mmol), MgCl2·6H2O (30 mmol) and

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MgSO4·7H2O (16 mmol) in 350 mL ultrapure water and subsequently adjusting the pH to 7.8

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with 0.1 M NaOH. This was diluted to required salinities during subsequent experiments.

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In a typical preparation, 250 µL of 40 nm AgNP stock suspension was sonicated for

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5 min in an ultrasonic bath (Bandelin Sonorex Digitec, 30 W output) to which was then added

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400 µL of BSA stock solution (250 µM) and mixed gently for 3 min Finally, 350 µL ASW

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stock solution was added, bringing the final volume to 1 mL and S·38, and the solution was

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mixed again. The samples were prepared and left uncovered in ambient conditions (22°C,

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natural light day-night cycle) until measurement. AgNPs and silver ions (Ag+) were separated

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by centrifugal ultrafiltration 29 in Whatman VectaSpin Micro centrifuge tubes through 12 kDa

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molecular weight cut-off cellulose triacetate membranes (GE Healthcare Life Sciences,

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Scherty, TX, USA). Centrifugation was carried out on an Eppendorf Centrifuge 5417R

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(Eppendorf, Hamburg, Germany) and samples were spun at 10000 × g for 20 min at 22°C.

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There was no evidence of AgNPs in the filtrate by UV absorption spectroscopy or dynamic

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light scattering (DLS).

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A double-beam Shimadzu UV-1800 spectrophotometer was used for measuring

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absorption spectra in the wavelength range 300-800 nm with 1 nm resolution. The samples

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were held in quartz glass cuvettes with an optical path length of 10 mm and measured in

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triplicate. Data processing was carried out on UVProbe 2.3.1. (Shimadzu, Kyoto, Japan) and

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Origin 9.0 (OriginLab Corporation, Northampton, MA, USA) software.

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Size distribution and zeta (ζ) potential were determined by means of dynamic and

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electrophoretic light scattering using a photon correlator spectrophotometer equipped with a

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532 nm ‘green’ laser (Zetasizer Nano ZS, Malvern Instruments, UK). Intensity of scattered

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light was detected at the angle of 173°. For DLS measurements samples were held in

5




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polystyrol/polystyrene cuvettes with 10 mm optical path-length. The hydrodynamic diameter

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(dh) was obtained as a value at peak maximum of size volume distribution function. The ζ

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potential was calculated from the measured electrophoretic mobility by means of the Henry

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equation using the Smoluchowski approximation. Each sample was measured 10 times and

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the results were expressed as an average value. Data processing was done by Zetasizer

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software 6.32 (Malvern instruments). All measurements were conducted at 22 ± 0.1 °C.

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AgNP and Ag+ ion concentrations were measured in duplicate (

Effect of Protein Corona on Silver Nanoparticle Stabilization and Ion

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