Influence of Solution Chemistry and Soft Protein Coronas on the

Jan 26, 2016 - ABSTRACT: The influence of solution chemistry and soft protein coronas on the interactions between citrate-coated silver nanoparticles ...
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Influence of Solution Chemistry and Soft Protein Corona on the Interactions of Silver Nanoparticles with Model Biological Membranes Qiaoying Wang, Myunghee Lim, Xitong Liu, Zhiwei Wang, and Kai Loon Chen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b04694 • Publication Date (Web): 26 Jan 2016 Downloaded from http://pubs.acs.org on February 9, 2016

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Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Environmental Science & Technology

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Influence of Solution Chemistry and Soft Protein Corona on the

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Interactions of Silver Nanoparticles with Model Biological

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Membranes

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Environmental Science & Technology Revised: January 21, 2016

Qiaoying Wang,1, 2, † Myunghee Lim,2, ‡, † Xitong Liu,2 Zhiwei Wang,1 and Kai Loon Chen2, *

1

School of Environmental Science and Engineering, State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, 1239 Siping Road, Shanghai 200092, PR China 2

26 27 28 29 30 31 32 33 34 35

Department of Geography and Environmental Engineering, Johns Hopkins University, Baltimore, Maryland 21218-2686, USA



Current Address: Yeosu Joint Inter Agency Chemical Emergency Preparedness Center, 34, Yeosusandan-ro, Yeosu-si, Jeollanamdo, 59631, Republic of Korea †

Both authors contributed equally to this work.

* Corresponding author: Kai Loon Chen, E-mail: [email protected], Phone: (410) 516-7095. 1

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Abstract

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The influence of solution chemistry and soft protein corona on the interactions between citrate-

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coated silver nanoparticles (AgNPs) and model biological membranes was investigated by

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assembling supported lipid bilayers (SLBs) composed of zwitterionic 1,2-dioleoyl-sn-glycero-3-

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phosphocholine (DOPC) on silica crystal sensors in a quartz crystal microbalance with

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dissipation monitoring (QCM-D). Our results show that the deposition rates of AgNPs on

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unmodified silica surfaces increased with increasing electrolyte concentrations under neutral pH

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conditions.

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indicating that the deposition of AgNPs on model cell membranes was controlled by electrostatic

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interactions. In the presence of human serum albumin (HSA) proteins at both pH 7 and pH 2, the

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colloidal stability of AgNPs was considerably enhanced due to the formation of HSA soft corona

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surrounding the nanoparticles. At pH 7, the deposition of AgNPs on SLBs was suppressed in the

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presence of HSA due to steric repulsion between HSA-modified AgNPs and SLBs. In contrast,

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pronounced deposition of HSA-modified AgNPs on SLBs was observed at pH 2.

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observation was attributed to the reduction of electrostatic repulsion, as well as conformation

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changes of adsorbed HSA under low pH conditions, resulting in the decrease of steric repulsion

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between AgNPs and SLBs.

Similar trends were observed when AgNPs were deposited on SLBs, hence

This

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Introduction

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Silver nanoparticles (AgNPs) are employed in various commercial products due to their

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unique optical, electronic, and antibacterial properties.1-4 With the rapid rise in the production

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and use of nanosilver-containing products, the inevitable release of AgNPs into the environment

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has raised concerns regarding their potential impacts on the ecosystem and human health.5-7

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Benn and Westerhoff8 demonstrated in their study that both colloidal and ionic Ag can be

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released from fabrics (socks) that contain nanosilver under normal washing conditions.

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Numerous studies have also been carried out to investigate the environmental fate and transport,

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as well as ecological impacts, of AgNPs.9, 10

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While the toxicity of silver ions has been well reported, the mechanisms for the cytotoxicity

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of AgNPs are still not completely understood.11 One proposed mechanism suggests that once

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attached on cell membrane, AgNPs can penetrate the membrane, causing an increase in

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membrane permeability and disturbance of cell respiration, thus resulting in cell death.12 2

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Moreover, the attachment of AgNPs to the cell membrane will expose the cell to an elevated

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concentration of silver ions.13 Thus, the attachment of AgNPs to cell membranes is expected to

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be a critical initial process that precedes the toxicity pathways.14-16

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Based on existing nanotoxicity studies, the interactions between nanomaterials and cell

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membranes are affected by various parameters, such as the physicochemical properties of the

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nanoparticles,17 composition of media (or solution),18 and components of cell membranes.19 In

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order to determine the main factors that govern the interactions between nanomaterials and cell

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membranes, several studies have been conducted with the use of lipid bilayers as model cell

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membranes.20-23 Frost et al.20 investigated the interactions of graphene oxide with positively and

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negatively charged lipid membranes. In their study, they emphasized the important role of

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electrostatic interactions in controlling the adhesion of graphene oxide to the model

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membranes.20

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Recent research has revealed that once nanoparticles enter into the bloodstream or other

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biological fluids, they can be readily coated with proteins, fatty acids, lipids, and other

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biomolecules.24,

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influenced by the coverage of biomolecules on the nanoparticle surface.26, 27 Lesniak et al.28

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suggested that the adsorption of proteins on the nanoparticle surface strongly reduces

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nanoparticle adhesion in comparison to what was observed for the bare material. The presence

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of high concentrations of proteins may “protect” cells from the nanoparticle-associated cytotoxic

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effects.29 The adsorption of proteins, resulting in the formation of a protein “corona” around the

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nanoparticle, can potentially reduce the unspecific interactions between nanoparticles and the

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cell membrane, and subsequently suppress the cytotoxicity of nanomaterials.29, 30

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Consequently, the interactions at the “nano-bio” interface can be greatly

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The formation of protein corona has been reported for a number of nanomaterials, including

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silica,31, 32 polystyrene,33 and gold particles.34 In general, the corona is composed of “hard” and

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“soft” components. The hard corona refers to strongly-adsorbed proteins on the nanoparticle

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surface, while the soft corona refers to proteins that are associated with the hard corona via weak

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protein-protein interactions.33, 35 Therefore, the soft corona is the external surface of a protein-

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coated nanoparticle and is expected to come into contact with the cell membrane when the

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nanoparticle approaches a cell.

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nanoparticles and biological membranes have been reported,28, 36, 37 no systematic investigation

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has been conducted on the influence of solution pH, ionic composition, and biomolecules on the

Although several studies on the interactions between

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interactions between AgNPs and model cell membranes.

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In this study, we first investigated the deposition (or adhesion) and release behavior of

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citrate-coated AgNPs on silica surfaces with the use of a quartz crystal microbalance with

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dissipation monitoring (QCM-D). Since silica is ubiquitous in natural aquatic systems and

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subsurface environments, these initial experiments will elucidate the interactions of AgNPs with

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naturally-occurring silica surfaces. For our subsequent experiments, we modified the silica

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surfaces with supported lipid bilayers (SLBs) and examined the deposition behavior of AgNPs

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on the bilayers, which are used as models for biological membranes. The effects of solution

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chemistry and soft protein corona on the kinetics and reversibility of the deposition of AgNPs on

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model cell membranes were systematically examined. The results obtained in this study are

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expected to provide a better understanding of the processes involved in the interactions of

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AgNPs with cell membranes.

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Materials and Methods Silver Nanoparticle Synthesis and Characterization. 38

Citrate-coated AgNPs were

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prepared through Tollens reaction,

followed by the cleaning and re-suspension of the

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nanoparticles in a trisodium citrate solution, as reported in our previous publication.39 The

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nanoparticle and dissolved silver concentrations of the citrate-coated AgNP stock solutions were

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determined to be 4.6–8.9 mg/L and 0.06–0.38 mg/L, respectively, using atomic absorption

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spectroscopy (AAS). The hydrodynamic size of the citrate-coated AgNPs was determined to be

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49–65 nm through dynamic light scattering (DLS).

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measurements is provided in the Supporting Information (SI).

The detailed procedure for the AAS

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Preparation of HSA-Modified AgNPs. The human serum albumin (HSA, MW 66,478 Da,

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Sigma-Aldrich) protein was selected for this study because it is the most abundant protein in the

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body.40, 41 For instance, HSA is present in blood serum and urine at concentrations of ca. 35–50

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g/L and 2.2–25 mg/L, respectively, while its concentration in saliva is typically < 0.5 g/L.40 The

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citrate-coated AgNPs and HSA were mixed at a number ratio of 1:10,000 in low-protein binding

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Eppendorf Protein LoBind tubes (Eppendorf®, America) and the suspension was equilibrated in

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an orbital shaker at 125 rpm and at 25 ºC for 2 h. The final suspension after this incubation

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process will be referred to as HSA-modified AgNPs. 4

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Preparation of Vesicle Stock Suspensions.

Zwitterionic 1,2-dioleoyl-sn-glycero-3-

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phosphocholine (DOPC, purity 99%) stock mixture (in chloroform) was purchased from Avanti

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Polar Lipids, Inc. (Alabaster, AL). The extrusion method was used to prepare unilamellar DOPC

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vesicle stock suspensions.42, 43 The detailed procedure is described in the SI. The hydrodynamic

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diameter of the vesicles was determined to be 102–115 nm through DLS measurements.

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Solution Chemistry. NaCl and CaCl2 stock solutions were prepared and filtered using 0.1

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µm PVDF syringe filters (Millipore, MA). NaHCO3 (0.15 mM) and HCl were used to adjust the

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pH to 7 and 2, respectively.

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Electrophoretic Mobility Measurements.

The influence of HSA soft corona on the

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electrokinetic properties of AgNPs was investigated through electrophoretic mobility (EPM)

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measurements. Suspensions of citrate-coated and HSA-modified AgNPs were prepared at an

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AgNP concentration of 4 mg/L and their EPMs were measured (ZetaPALS, Brookhaven

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Instruments Corp., Holtsville, NY) as a function of NaCl concentration at both pH 7 and 2. For

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most conditions, ten measurements were conducted. Five measurements were conducted at 50

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and 70 mM NaCl to minimize the effect of AgNP aggregation.

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Aggregation of AgNPs. To examine the aggregation behavior of citrate-coated and HSA-

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modified AgNPs, time-resolved DLS measurements were performed with a light scattering unit

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using the method described in our previous publication.39 The detailed procedure for the DLS

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measurements is also provided in the SI.

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Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) Measurements.

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The interactions of AgNPs with silica surfaces and DOPC SLBs were investigated using a QCM-

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D (Q-Sense E4, Västra Frölunda, Sweden). Piezoelectric quartz crystal sensors with silica-

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coated surfaces (QSX 303, Q-Sense) were used for the QCM-D measurements. The frequency

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and dissipation responses of the crystals at multiple harmonics (n = 1, 3, 5, 7, 9, 11, and 13) were

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monitored continuously and the data at the 3rd harmonic was presented in this study. All the

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QCM-D measurements were performed under a constant flow rate (0.1 mL/min) at 25 °C. The

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electrolyte solutions were degassed for 10 min in an ultrasonication bath (Branson 5510RDTH,

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output power 135 W, frequency 40 kHz) before use. Before the QCM-D experiments, all the

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flow modules were thoroughly rinsed with a surfactant solution (1% Hellmanex II, Hellma

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GmbH & Co. KG, Germany) and DI water. The silica-coated crystal sensors were soaked in the

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surfactant solution for 30 min, rinsed with deionized (DI) water (Millipore, MA), dried with 5

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nitrogen stream, and oxidized in a UV-ozone chamber (Procleaner 110, BioForce Nanosciences,

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Inc., Ames, IA) for 20 min. Two flow modules were used to produce duplicate data for each

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experimental condition.

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The deposition experiments were performed either on silica-coated crystal sensors or on

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sensors that are modified with DOPC SLBs using the method reported in our previous

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publications.22, 44, 45 To assemble a continuous DOPC SLB on a sensor, DOPC vesicles were

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injected into QCM-D system and deposited on the crystal sensor. When the vesicular coverage

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on the crystal surface was high enough to trigger the rupture of the deposited vesicles, a

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continuous SLB was formed on the crystal surface.42 The encapsulated solution within the

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ruptured vesicles was released and resulted in a decrease in deposited mass and, hence, an

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increase in frequency. The representative frequency shifts during the formation of a DOPC SLB

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on a silica surface at pH 7 were shown in SI Figure S1. The formation of a SLB was verified by

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the overall frequency decrease of 24.5 ± 0.5 Hz, which is the same as that in previous studies.22,

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42, 43

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Specific steps for the assembly of a SLB are the sequential introduction of 5 mL of 10 mM

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N-(2-hydroxyethyl)-piperazine-N’-(2-ethanesulfonic acid) (HEPES) buffer prepared in a 150

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mM NaCl solution, 5 mL of 0.07 g/L DOPC vesicle suspension prepared in HEPES buffer, and

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another 5 mL of HEPES buffer into the flow module.42, 46 The flow module was then flushed

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with the electrolyte solution of interest. Subsequently, the citrate-coated or HSA-modified

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AgNP suspension (4 mg/L AgNPs) was prepared in the same solution chemistry and introduced

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into the flow module. The deposition kinetics of AgNPs on silica surfaces or DOPC SLBs were

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quantified using the attachment efficiency, α, which can be understood as the probability of an

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attachment resulting from an AgNP approaching the silica surface or SLB.47 The attachment

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efficiency was calculated by normalizing the rate of frequency shift during AgNP deposition on

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silica surfaces or SLBs to the rate of frequency shift during favorable deposition at the same

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solution chemistry.22,

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experiments were performed on the silica surfaces modified with positively charged poly-L-

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lysine (PLL).23,

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investigated by sequentially flushing the crystal with different electrolyte solutions of interest.

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The flushing process was stopped when the shift in the normalized frequency at the 3rd overtone,

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∆F, was less than 0.2 Hz within a time period of 10 min.48, 49

44

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To obtain the favorable deposition rates of AgNPs, the deposition

For the release experiments, the reversibility of AgNPs deposition was

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Results and Discussion

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Deposition Kinetics of Citrate-Coated AgNPs on Silica Surfaces. The deposition rates

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of citrate-coated AgNPs on silica surfaces at pH 7 are presented as functions of NaCl and CaCl2

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concentrations in Figure 1a and b, respectively. The deposition profiles of AgNPs on silica

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surfaces at pH 7 at selected NaCl concentrations was shown in SI Figure S2. For both NaCl and

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CaCl2, the deposition of AgNPs on silica occurred slowly under low electrolyte concentrations.

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As the electrolyte concentration was raised, the deposition rates increased and reached a

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maximum at ca. 200 mM NaCl and 3 mM CaCl2. When the NaCl and CaCl2 concentrations

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exceeded 200 mM and 3 mM, respectively, the deposition rates reached a plateau.

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FIGURE 1 Figure 1c and d show the attachment efficiencies of citrate-coated AgNPs on silica surfaces

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as functions of NaCl and CaCl2 concentrations, respectively, at pH 7.

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efficiencies are calculated by normalizing the deposition rates on silica surfaces to the favorable

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deposition rates on PLL-modified surfaces (SI Figure S3).

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concentrations, the attachment efficiency profiles can be classified into unfavorable (α < 1) and

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favorable (α = 1) deposition regimes. In the unfavorable regime, the attachment efficiency

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increased with the rise in electrolyte concentrations. This increase in deposition kinetics is due

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to charge screening (NaCl) or charge neutralization (CaCl2) effects which resulted in a decrease

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in the energy barrier.50 In the favorable regime, the surface charge of AgNPs and silica surfaces

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were completely screened (or neutralized in the presence of CaCl2), such that the interactions

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between AgNPs and silica surfaces are dominated by van der Waals attraction. By extrapolating

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through the unfavorable and favorable regimes and determining the intersection of the two

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extrapolations, the critical deposition concentration (CDC), or the minimum electrolyte

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concentration that allows for favorable deposition to take place,48, 51 can be obtained. Using this

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approach, the intersections of the extrapolations (dashed lines in Figure 1c and d) yielded CDCs

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of 272.8 mM NaCl and 3.2 mM CaCl2. The CDC in CaCl2 was much smaller than that in NaCl

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because Ca2+ ions are much more effective in screening surface charges than Na+ ions.50

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Additionally, Ca2+ ions can neutralize the surface charge of citrate-coated AgNPs through

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complex formation with the carboxyl groups of the adsorbed citrate molecules.52 In our previous

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study,39 we verified that the EPMs of citrate-coated AgNPs were considerably less negative in 7

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For both NaCl and CaCl2

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the presence of CaCl2 than in NaCl, which demonstrates that Ca2+ ions are more efficient in

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reducing the energy barrier than Na+ ions. In the same study,39 we also showed that the critical

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coagulation concentration of the citrate-coated AgNPs is 2.1 mM CaCl2. Thus, the charges of

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the AgNPs are expected to be completely neutralized at 3 mM CaCl2.

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Release of Deposited Citrate-Coated AgNPs from Silica Surfaces. To investigate the

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reversibility of the deposition of AgNPs on silica surfaces, the AgNPs were first deposited on

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silica-coated crystals at either 300 mM NaCl or 3 mM CaCl2. These concentrations were

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selected in order to allow for favorable deposition. In order to evaluate the reversibility of AgNP

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deposition, the deposited AgNPs were first rinsed with the same electrolyte solution (300 mM

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NaCl or 3 mM CaCl2) and then with low ionic strength solutions. Figure 2a shows that the

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frequency decreased from –6 Hz to –14 Hz when the deposition of AgNPs took place at 300 mM

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NaCl (from 62 to 142 min) and no release was observed when the deposited AgNPs were rinsed

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with a 300 mM NaCl solution (from 142 to 182 min). When the deposited AgNPs were rinsed

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with a 1 mM NaCl solution (from 182 min onwards), the frequency increased to ca. –1 Hz

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instantaneously, indicating that almost all the deposited AgNPs were released from the silica

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surface. The deposition and release experiment in the presence of CaCl2 was conducted using a

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similar approach (Figure 2b). AgNPs were first deposited on the silica surface at 3 mM CaCl2

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(from 40 to 150 min), during which the frequency decreased from –0.2 Hz to –5 Hz. The

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deposited AgNPs were then subsequently rinsed with 3 mM CaCl2, 1 mM CaCl2, and 1 mM

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NaCl eluents, during which slight frequency shifts occurred likely due to the switch of

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background solutions.22, 48 This result showed that no release of AgNPs occurred upon exposure

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to low ionic strength solutions.

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FIGURE 2

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According to the Derjaguin–Landau–Verwey–Overbeek (DLVO) theory,50 a reduction in

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NaCl concentration from 300 to 1 mM will reduce the charge screening effect, which in turn may

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result in a significant rise in the electrostatic repulsion between the deposited AgNPs and silica

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surface.48, 49, 53 Therefore, almost all of the deposited AgNPs were released from silica upon

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exposure to 1 mM NaCl (Figure 2a). However, in the presence of CaCl2 (Figure 2b), the surface

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charge of citrate-coated AgNPs was neutralized through the formation of calcium citrate

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complexes.52, 54 In order for the AgNPs that were deposited in the presence of calcium to be

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released, the calcium citrate complexes have to be dissociated such that the surface charge of 8

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citrate-coated AgNPs can be restored. However, due to the strong chelating ability of citrate to

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Ca2+ ions, we speculate that the degree of Ca2+ dissociation was low.55 Therefore, the rinsing

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with eluents of low CaCl2 and NaCl concentrations did not result in a significant release of the

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deposited AgNPs.

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Deposition Kinetics of Citrate-Coated AgNPs on DOPC SLBs. The deposition rates of

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citrate-coated AgNPs on DOPC SLBs at pH 7 are presented as a function of NaCl concentration

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in Figure 3a.

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concentration was increased from 16 to 300 mM. The attachment efficiencies for the deposition

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of AgNPs on DOPC SLBs are presented as a function of NaCl concentration in Figure 3b.

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According to our previous study,22 the DOPC lipid bilayers were negatively charged at pH 7 and

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the magnitude of EPMs decreased with increasing ionic strength. The trend for the attachment

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efficiencies on SLBs is similar to that for deposition on silica surfaces (Figure 1c and d).

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Specifically, unfavorable and favorable deposition regimes were observed for the deposition of

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AgNPs on SLBs, thus indicating that the deposition kinetics on the model membranes were

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controlled by electrostatic interactions. By extrapolating through the unfavorable and favorable

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regimes, the CDC in NaCl for the deposition of AgNPs on DOPC SLBs is determined to be 70

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mM.

The deposition rates of AgNPs on DOPC SLBs increased when the NaCl

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FIGURE 3

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Release of Deposited Citrate-Coated AgNPs from DOPC SLBs. To investigate the

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reversibility of AgNP deposition on DOPC SLBs, the SLBs were first assembled on the silica-

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coated crystals.

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experiments were conducted as discussed for silica surfaces. Figure 3c shows the frequency

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shifts during the formation of a DOPC SLB followed by the deposition and release of AgNPs on

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the SLB in the presence of 300 mM NaCl at pH 7. Figure 3d shows the close-up of the

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frequency shifts during the deposition and release of AgNPs on the bilayer. The frequency

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decreased from –32.6 to –38.1 Hz (i.e., a decrease of 5.5 Hz) when the AgNPs were deposited on

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the SLB at 300 mM NaCl (from 80 to 142 min). No release was observed when the deposited

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AgNPs were rinsed at 300 mM NaCl (from 142 to 170 min). When the deposited AgNPs were

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rinsed with a 1 mM NaCl solution for 40 min (from 170 min onwards), the frequency increased

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to –27.5 Hz, hence resulting in an overall frequency increase of 10.6 Hz. In order to account for

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the frequency shift caused by the change in background solutions (or buffer effect),22,

AgNPs were deposited on the SLBs at 300 mM NaCl and the release

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the

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experiment with same electrolyte solutions was performed in the absence of AgNPs. It was

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shown (SI Figure S4) that the transition from 300 to 1 mM NaCl resulted in an increase of 7.0 Hz.

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Therefore, the rise in frequency resulting only from the release of AgNPs is 3.6 Hz, implying

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that ca. 65% of deposited AgNPs were released upon exposure to 1 mM NaCl (Figure 3d). The

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release of deposited AgNPs from model cell membranes was higher than that for multiwalled

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carbon nanotubes (ca. 20%), as observed in our earlier study.22 An implication of this finding is

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that AgNPs deposited in the human gastrointestinal tract may be released when low-ionic-

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strength fluids (e.g., water) are ingested.

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Additionally, the release of AgNPs deposited on the SLBs at 1 mM CaCl2 was investigated.

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The deposited AgNPs were rinsed with 1 mM CaCl2 followed by 1 mM NaCl eluents. Similar to

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the observations for AgNPs deposited on silica surfaces, slight frequency shifts occurred during

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the rinse with the eluents (SI Figure S5), indicating that not many AgNPs were released from the

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SLBs, probably due to the strong chelating ability of citrate to Ca2+ ions.

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Influence of Soft Corona on Electrokinetic Properties of AgNPs. The EPMs of citrate-

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coated and HSA-modified AgNPs at pH 7 are presented as functions of NaCl concentration in

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Figure 4a. Under neutral pH conditions, both citrate-coated and HSA-modified AgNPs were

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negatively charged. The EPMs of both citrate-coated and HSA-modified AgNPs became less

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negative with increasing NaCl concentrations due to an increase in charge screening effect.

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Since the pKa values of citric acid are 3.13, 4.72, and 6.33,56 the carboxylic acid groups of citrate

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molecules are mostly deprotonated at pH 7 and thus the citrate-coated AgNPs were negatively

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charged. Since HSA has both carboxyl and amino groups and the isoelectric point (IEP) of HSA

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is ca. 5,57 HSA was negatively charged at pH 7. Even though the AgNPs and HSA were both

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negatively charged, HSA can still adsorb on the AgNPs through hydrogen bonding.24

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FIGURE 4

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Since some biological media have low pH (e.g., pH of gastric fluid = 1.12),58 the influence

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of soft protein corona on the electrokinetic properties of AgNPs at pH 2 was also investigated.

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The EPMs of citrate-coated and HSA-modified AgNPs at pH 2 are presented as functions of

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NaCl concentration in Figure 4b. While the citrate-coated AgNPs were negatively charged at pH

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2, the HSA-modified AgNPs were positively charged. At pH 2, HSA was positively charged

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since the carboxyl and ammonium groups of HSA were fully protonated.57 Thus, under such low

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pH conditions, the oppositely charged proteins and nanoparticle surfaces exhibited strong 10

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affinity59 and the adsorption of HSA on the AgNPs resulted in the reversal of nanoparticle

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surface charge from negative to positive. Additionally, it is observed that the magnitude of the

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EPMs for both citrate-coated and HSA-modified AgNPs decreased with increasing NaCl

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concentrations due to charge screening effects.

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Influence of Soft Corona on Aggregation Behavior of AgNPs. The aggregation profiles

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of citrate-coated and HSA-modified AgNPs in the presence of 150 mM NaCl (ionic strength of

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biological fluids) at pH 7 and pH 2 are presented in Figure 4c and d, respectively. At both pH

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conditions, the hydrodynamic diameter of citrate-coated AgNPs increased from 60 nm to ca. 175

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nm after a time period of 45 min, whereas the size of HSA-modified AgNPs increased to 75 nm

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and 100 nm at pH 7 and pH 2, respectively. The aggregation rate of HSA-modified AgNPs was

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slower than that of citrate-coated AgNPs for both pH conditions, indicating that the addition of

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HSA can stabilize the AgNP suspensions. At pH 7, both citrate-coated and HSA-modified

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AgNPs were negatively charged and their EPMs were very similar (Figure 4a). Therefore, rather

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than electrostatic repulsion, the elevated stability of AgNPs was attributed to the steric repulsion

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imparted by the soft HSA coronas. Steric effects were also proposed for TiO2 colloids in HSA.60

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At pH 2, the HSA-modified AgNPs were positively charged due to the adsorbed HSA.

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Based on the trends shown in Figure 4b, the magnitudes of the EPMs of HSA-modified and

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citrate-coated AgNPs are likely to be close to zero at 150 mM NaCl. Despite the similar

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mobilities of both AgNPs, the aggregation rate of HSA-modified AgNPs was lower than that of

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citrate-coated AgNPs. The reduction in the aggregation kinetics in the presence of HSA was

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hence attributed to the steric repulsion between the nanoparticles, similar to the case at pH 7.

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We noted that the aggregation rates of citrate-coated AgNPs at pH 7 and 2 were very similar

336

in the presence of 150 mM NaCl (SI Figure S6a), likely due to the dominance of van der Waals

337

interactions at such a high NaCl concentration.

338

aggregation rate of HSA-modified AgNPs at pH 2 was slightly higher than that at pH 7 (SI

339

Figure S6b). It is known that HSA is composed of three homologous domains, each of which

340

displays specific structural and functional characteristics.41,

341

transitions in the acidic pH region (pH < 3.5).41, 61, 62 When the pH was decreased to 2, a marked

342

reduction of helix content in HSA was observed.61, 63 This conformation change of HSA was

343

speculated to result in a drop in steric repulsion between the HSA-modified AgNPs and hence in

344

the faster aggregation of HSA-modified AgNPs at pH 2.

Interestingly, it was observed that the

61

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HSA can undergo structural

Additionally, due to the lower

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345

magnitude of EPMs of HSA-modified AgNPs at pH 2, the reduced electrostatic repulsion was

346

expected to facilitate the aggregation of HSA-modified AgNPs.

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347

Influence of Soft Corona on Deposition of AgNPs on DOPC SLBs. Deposition profiles

348

of HSA proteins, citrate-coated AgNPs, and HSA-modified AgNPs on DOPC SLBs at pH 7 and

349

2 (all at 150 mM NaCl) are presented in Figure 5. At pH 7 (Figure 5a), very small frequency

350

shifts were observed when the HSA proteins and HSA-modified AgNPs were introduced into the

351

QCM-D modules, indicating that slight deposition of HSA proteins and HSA-modified AgNPs

352

took place.

353

observed (from 0 to –4 Hz within 30 min), indicating that considerable deposition occurred at pH

354

7. Lesniak et al.28 reported the similar results when investigated the effects of hard protein

355

corona on the adhesion of carboxylated polystyrene nanoparticles on 2-oleoyl-1-palmitoyl-sn-

356

glycero-3-phosphocholine (POPC) lipid bilayers. For bare nanoparticles, they observed a strong

357

adsorption for all concentrations. In contrast, for hard corona-covered nanoparticles, little to no

358

adsorption occurs even for the highest nanoparticle concentration.

For the citrate-coated AgNPs, in contrast, a sharp decrease in frequency was

FIGURE 5

359 360

Since the IEP of DOPC is ca. 4,

64

DOPC SLBs are negatively charged at pH 7. Although

361

the citrate-coated AgNPs were also negatively charged, they underwent relatively fast deposition

362

on the DOPC SLBs due to the considerable screening of surface charge of the AgNPs and

363

bilayers at 150 mM NaCl. Conversely, in the presence of HSA proteins, the formation of HSA

364

coronas around the AgNPs lowered the surface energy of the nanoparticles and reduced the

365

nonspecific interactions between the HSA-modified AgNPs and SLBs.28 Additionally, steric

366

repulsion between HSA-modified AgNPs and SLBs may inhibit the deposition of the AgNPs on

367

SLBs. Therefore, no considerable deposition of HSA-modified AgNPs on SLBs was observed at

368

pH 7.

369

In contrast, it is shown in Figure 5b that considerable deposition of both citrate-coated and

370

HSA-modified AgNPs on SLBs occurred at pH 2. Since the EPMs of both nanoparticles are

371

close to zero at pH 2 and high ionic strength conditions (Figure 4b), van der Waals attraction

372

between the nanoparticles and SLBs is expected to dominate over electrostatic repulsion under

373

such conditions.

374

occurred under the low pH condition and resulted in a drop of steric repulsion between the

375

AgNPs and SLBs, thus allowing for the deposition HSA-modified AgNPs on SLBs.

Additionally, the conformational change of the HSA coronas may have

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Implications.

In order to investigate the toxicity of AgNPs in environmental and

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biological systems, this study evaluated the influence of solution chemistry and soft protein

378

corona on the interactions between AgNPs and model biological membranes. Our results show

379

that the electrolyte concentration, cation valence, and protein corona play important roles in

380

controlling the deposition and release of AgNPs on silica surfaces and model cell membranes.

381

The HSA proteins that adsorbed onto nanoparticle surfaces significantly affected the

382

nanoparticle electrokinetic properties, aggregation behavior, and attachment efficiency of AgNPs

383

to model biological membranes. Our results show that, at neutral pH conditions, the presence of

384

soft protein corona resulted in a reduction in the attachment of AgNPs to lipid bilayers. This

385

observation possibly implies that when AgNPs enter the bloodstream65 (pH of ca. 7.4),66 HSA in

386

the blood serum may inhibit the direct contact of AgNPs with red blood cells and potentially

387

mitigate the hemolytic activity of the AgNPs.67 We also observed that protein-coated AgNPs

388

underwent faster attachment to lipid bilayers at pH 2 than at pH 7. Therefore, in acidic gastric

389

fluids, the formation of protein corona may enhance AgNP attachment to cell membranes. In

390

addition to proteins, the adsorption of other biomolecules, such as fatty acids and lipids from

391

biological fluids, can result in the formation of coronas around the nanoparticle.68 Given the

392

complex mechanisms for the formation of coronas and the numerous possible combinations of

393

nanoparticle-biomolecule complexes, further studies on the influence of biomolecule coronas on

394

nanoparticle−membrane interactions will provide more insights into the cytotoxicity of

395

nanoparticles in environmental and biological systems.

396 397

Acknowledgements

398

This research was funded by the Semiconductor Research Corporation (award 425-MC-2001,

399

project 425.041), National Natural Science Foundation of China (51308400), and China

400

Postdoctoral Science Foundation (2013M540389 & 2014T70429).

401

postdoctoral fellowship from the National Research Foundation of Korea (Ministry of Education,

402

Science and Technology) (NRF-2011-357-D00143).

M. L. acknowledges a

403 404

Supporting Information Available

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Additional figures and details for Materials and Methods are presented.

406

available free of charge via the Internet at http://pubs.acs.org. 13

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Literature Cited

408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453

1. Wu, W. J.; Wu, M. Z.; Sun, Z. Q.; Li, G.; Ma, Y. Q.; Liu, X. S.; Wang, X. F.; Chen, X. S., Morphology controllable synthesis of silver nanoparticles: Optical properties study and SERS application. J Alloy Compd 2013, 579, 117-123. 2. Chen, D. P.; Qiao, X. L.; Qiu, X. L.; Chen, J. G., Synthesis and electrical properties of uniform silver nanoparticles for electronic applications. J Mater Sci 2009, 44, (4), 1076-1081. 3. Khan, M.; Khan, S. T.; Khan, M.; Adil, S. F.; Musarrat, J.; Al-Khedhairy, A. A.; Al-Warthan, A.; Siddiqui, M. R. H.; Alkhathlan, H. Z., Antibacterial properties of silver nanoparticles synthesized using Pulicaria glutinosa plant extract as a green bioreductant. Int J Nanomed 2014, 9, 3551-3565. 4. Xie, C. M.; Lu, X.; Wang, K. F.; Meng, F. Z.; Jiang, O.; Zhang, H. P.; Zhi, W.; Fang, L. M., Silver Nanoparticles and Growth Factors Incorporated Hydroxyapatite Coatings on Metallic Implant Surfaces for Enhancement of Osteoinductivity and Antibacterial Properties. Acs Appl Mater Inter 2014, 6, (11), 8580-8589. 5. Teow, Y.; Asharani, P. V.; Hande, M. P.; Valiyaveettil, S., Health impact and safety of engineered nanomaterials. Chem Commun 2011, 47, (25), 7025-7038. 6. Lapresta-Fernandez, A.; Fernandez, A.; Blasco, J., Nanoecotoxicity effects of engineered silver and gold nanoparticles in aquatic organisms. Trac-Trend Anal Chem 2012, 32, 40-59. 7. Tejamaya, M.; Romer, I.; Merrifield, R. C.; Lead, J. R., Stability of Citrate, PVP, and PEG Coated Silver Nanoparticles in Ecotoxicology Media. Environmental science & technology 2012, 46, (13), 70117017. 8. Benn, T. M.; Westerhoff, P., Nanoparticle silver released into water from commercially available sock fabrics. Environmental science & technology 2008, 42, (11), 4133-4139. 9. Love, S. A.; Maurer-Jones, M. A.; Thompson, J. W.; Lin, Y. S.; Haynes, C. L., Assessing Nanoparticle Toxicity. Annu Rev Anal Chem 2012, 5, 181-205. 10. Reidy, B.; Haase, A.; Luch, A.; Dawson, K. A.; Lynch, I., Mechanisms of Silver Nanoparticle Release, Transformation and Toxicity: A Critical Review of Current Knowledge and Recommendations for Future Studies and Applications. Materials 2013, 6, (6), 2295-2350. 11. Dubey, P.; Matai, I.; Kumar, S. U.; Sachdev, A.; Bhushan, B.; Gopinath, P., Perturbation of cellular mechanistic system by silver nanoparticle toxicity: Cytotoxic, genotoxic and epigenetic potentials. Advances in colloid and interface science 2015, 221, 4-21. 12. Shahverdi, A. R.; Fakhimi, A.; Shahverdi, H. R.; Minaian, S., Synthesis and effect of silver nanoparticles on the antibacterial activity of different antibiotics against Staphylococcus aureus and Escherichia coli. Nanomed-Nanotechnol 2007, 3, (2), 168-171. 13. Khanh An, H.; McCaffery, J. M.; Chen, K. L., Heteroaggregation Reduces Antimicrobial Activity of Silver Nanoparticles: Evidence for Nanoparticle Cell Proximity Effects (vol 1, pg 361, 2014). Environmental Science & Technology Letters 2015, 2, (1), 19-19. 14. Bondarenko, O.; Ivask, A.; Kakinen, A.; Kurvet, I.; Kahru, A., Particle-Cell Contact Enhances Antibacterial Activity of Silver Nanoparticles. Plos One 2013, 8, (5). 15. McQuillan, J. S.; Infante, H. G.; Stokes, E.; Shaw, A. M., Silver nanoparticle enhanced silver ion stress response in Escherichia coli K12. Nanotoxicology 2012, 6, (8), 857-866. 16. Chen, K. L.; Bothun, G. D., Nanoparticles Meet Cell Membranes: Probing Nonspecific Interactions. using Model Membranes. Environmental science & technology 2014, 48, (2), 873-880. 17. Jiang, W.; Kim, B. Y. S.; Rutka, J. T.; Chan, W. C. W., Nanoparticle-mediated cellular response is size-dependent. Nature nanotechnology 2008, 3, (3), 145-150. 18. Teeguarden, J. G.; Hinderliter, P. M.; Orr, G.; Thrall, B. D.; Pounds, J. G., Particokinetics in vitro: Dosimetry considerations for in vitro nanoparticle toxicity assessments. Toxicological Sciences 2007, 95, (2), 300-312. 14

ACS Paragon Plus Environment

Page 15 of 23

454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499

Environmental Science & Technology

19. Hook, F.; Kasemo, B.; Grunze, M.; Zauscher, S., Quantitative Biological Surface Science: Challenges and Recent Advances. Acs Nano 2008, 2, (12), 2428-2436. 20. Frost, R.; Jonsson, G. E.; Chakarov, D.; Svedhem, S.; Kasemo, B., Graphene Oxide and Lipid Membranes: Interactions and Nanocomposite Structures. Nano letters 2012, 12, (7), 3356-3362. 21. Cho, N. J.; Jackman, J. A.; Liu, M.; Frank, C. W., pH-Driven Assembly of Various Supported Lipid Platforms: A Comparative Study on Silicon Oxide and Titanium Oxide. Langmuir : the ACS journal of surfaces and colloids 2011, 27, (7), 3739-3748. 22. Yi, P.; Chen, K. L., Interaction of Multiwalled Carbon Nanotubes with Supported Lipid Bilayers and Vesicles as Model Biological Membranes. Environmental science & technology 2013, 47, (11), 57115719. 23. Hou, W. C.; Moghadam, B. Y.; Corredor, C.; Westerhoff, P.; Posner, J. D., Distribution of functionalized gold nanoparticles between water and lipid bilayers as model cell membranes. Environ Sci Technol 2012, 46, (3), 1869-76. 24. Chen, R.; Choudhary, P.; Schurr, R. N.; Bhattacharya, P.; Brown, J. M.; Ke, P. C., Interaction of lipid vesicle with silver nanoparticle-serum albumin protein corona. Appl Phys Lett 2012, 100, (1). 25. Lynch, I.; Salvati, A.; Dawson, K. A., PROTEIN-NANOPARTICLE INTERACTIONS What does the cell see? Nature nanotechnology 2009, 4, (9), 546-547. 26. Ge, C. C.; Du, J. F.; Zhao, L. N.; Wang, L. M.; Liu, Y.; Li, D. H.; Yang, Y. L.; Zhou, R. H.; Zhao, Y. L.; Chai, Z. F.; Chen, C. Y., Binding of blood proteins to carbon nanotubes reduces cytotoxicity. Proceedings of the National Academy of Sciences of the United States of America 2011, 108, (41), 16968-16973. 27. Drescher, D.; Orts-Gil, G.; Laube, G.; Natte, K.; Veh, R. W.; Osterle, W.; Kneipp, J., Toxicity of amorphous silica nanoparticles on eukaryotic cell model is determined by particle agglomeration and serum protein adsorption effects. Anal Bioanal Chem 2011, 400, (5), 1367-1373. 28. Lesniak, A.; Salvati, A.; Santos-Martinez, M. J.; Radomski, M. W.; Dawson, K. A.; Aberg, C., Nanoparticle Adhesion to the Cell Membrane and Its Effect on Nanoparticle Uptake Efficiency. Journal of the American Chemical Society 2013, 135, (4), 1438-1444. 29. Kim, J. A.; Salvati, A.; Aberg, C.; Dawson, K. A., Suppression of nanoparticle cytotoxicity approaching in vivo serum concentrations: limitations of in vitro testing for nanosafety. Nanoscale 2014, 6, (23), 14180-14184. 30. Monopoli, M. P.; Aberg, C.; Salvati, A.; Dawson, K. A., Biomolecular coronas provide the biological identity of nanosized materials. Nature nanotechnology 2012, 7, (12), 779-786. 31. Monopoli, M. P.; Walczyk, D.; Campbell, A.; Elia, G.; Lynch, I.; Bombelli, F. B.; Dawson, K. A., Physical-Chemical Aspects of Protein Corona: Relevance to in Vitro and in Vivo Biological Impacts of Nanoparticles. Journal of the American Chemical Society 2011, 133, (8), 2525-2534. 32. Lesniak, A.; Fenaroli, F.; Monopoli, M. R.; Aberg, C.; Dawson, K. A.; Salvati, A., Effects of the Presence or Absence of a Protein Corona on Silica Nanoparticle Uptake and Impact on Cells. Acs Nano 2012, 6, (7), 5845-5857. 33. Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K. A., Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proceedings of the National Academy of Sciences of the United States of America 2008, 105, (38), 1426514270. 34. Maiorano, G.; Sabella, S.; Sorce, B.; Brunetti, V.; Malvindi, M. A.; Cingolani, R.; Pompa, P. P., Effects of Cell Culture Media on the Dynamic Formation of Protein-Nanoparticle Complexes and Influence on the Cellular Response. Acs Nano 2010, 4, (12), 7481-7491. 35. Walkey, C. D.; Chan, W. C. W., Understanding and controlling the interaction of nanomaterials with proteins in a physiological environment. Chemical Society reviews 2012, 41, (7), 2780-2799.

15

ACS Paragon Plus Environment

Environmental Science & Technology

500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545

36. Bailey, C. M.; Kamaloo, E.; Waterman, K. L.; Wang, K. F.; Nagarajan, R.; Camesano, T. A., Size dependence of gold nanoparticle interactions with a supported lipid bilayer: A QCM-D study. Biophys Chem 2015, 203, 51-61. 37. Yang, C.; Xie, H.; Li, Q.-C.; Sun, E.-J.; Su, B.-L., Adherence and interaction of cationic quantum dots on bacterial surfaces. Journal of colloid and interface science 2015, 450, 388-395. 38. Panacek, A.; Kvitek, L.; Prucek, R.; Kolar, M.; Vecerova, R.; Pizurova, N.; Sharma, V. K.; Nevecna, T.; Zboril, R., Silver colloid nanoparticles: Synthesis, characterization, and their antibacterial activity. J Phys Chem B 2006, 110, (33), 16248-16253. 39. Huynh, K. A.; Chen, K. L., Aggregation kinetics of citrate and polyvinylpyrrolidone coated silver nanoparticles in monovalent and divalent electrolyte solutions. Environ Sci Technol 2011, 45, (13), 556471. 40. Wang, R. S. E.; Tian, L.; Chang, Y. H., A homogeneous fluorescent sensor for human serum albumin. J Pharmaceut Biomed 2012, 63, 165-169. 41. Peters, T., All about Albumin. Academic Press: New York, 1996. 42. Richter, R.; Mukhopadhyay, A.; Brisson, A., Pathways of lipid vesicle deposition on solid surfaces: A combined QCM-D and AFM study. Biophys J 2003, 85, (5), 3035-3047. 43. Cho, N. J.; Frank, C. W.; Kasemo, B.; Hook, F., Quartz crystal microbalance with dissipation monitoring of supported lipid bilayers on various substrates. Nature protocols 2010, 5, (6), 1096-1106. 44. Liu, X.; Chen, K. L., Interactions of Graphene Oxide with Model Cell Membranes: Probing Nanoparticle Attachment and Lipid Bilayer Disruption. Langmuir : the ACS journal of surfaces and colloids 2015, 31, (44), 12076-12086. 45. Liu, X.; Chen, K. L., Aggregation and interactions of chemical mechanical planarization nanoparticles with model biological membranes: role of phosphate adsorption. Environmental Science: Nano 2016. 46. Richter, R. P.; Him, J. L. K.; Tessier, B.; Tessier, C.; Brisson, A. R., On the kinetics of adsorption and two-dimensional self-assembly of annexin A5 on supported lipid bilayers. Biophys J 2005, 89, (5), 33723385. 47. Yao, K. M.; Habibian, M. M.; Omelia, C. R., Water and Waste Water Filtration. Concepts and Applications. Environmental science & technology 1971, 5, (11), 1105-1112. 48. Yi, P.; Chen, K. L., Influence of surface oxidation on the aggregation and deposition kinetics of multiwalled carbon nanotubes in monovalent and divalent electrolytes. Langmuir 2011, 27, (7), 3588-99. 49. Yi, P.; Chen, K. L., Release Kinetics of Multiwalled Carbon Nanotubes Deposited on Silica Surfaces: Quartz Crystal Microbalance with Dissipation (QCM-D) Measurements and Modeling. Environmental science & technology 2014, 48, (8), 4406-4413. 50. Elimelech, M. G., J.; Jia, X.; Williams, R. A. , Particle Deposition and Aggregation-Measurement, Modelling and Simulation. Butterworth-Heinemann: Oxford, England, 1995. 51. Chen, K. L.; Elimelech, M., Interaction of Fullerene (C-60) Nanoparticles with Humic Acid and Alginate Coated Silica Surfaces: Measurements, Mechanisms, and Environmental Implications. Environmental science & technology 2008, 42, (20), 7607-7614. 52. Pearce, K. N., Formation-Constants for Magnesium and Calcium Citrate Complexes. Aust J Chem 1980, 33, (7), 1511-1517. 53. Chen, K. L.; Elimelech, M., Aggregation and deposition kinetics of fullerene (C-60) nanoparticles. Langmuir : the ACS journal of surfaces and colloids 2006, 22, (26), 10994-11001. 54. Ou, Y.; Chen, B.; Peng, H.; Gui, B. S.; Yao, X. Q.; Ouyang, J. M., Thermodynamic Features of Diethyl Citrate Calcium Complexes and Factors Affecting the Complex Stability. Asian J Chem 2012, 24, (10), 4717-4722.

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Page 17 of 23

546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577

Environmental Science & Technology

55. Martell, A. E.; Smith, R. M.; Motekaitis, R., NIST critically selected stability constants of metal complexes. NIST standard reference database 2004, 46, (6.0). 56. Benjamin, M. M., Water Chemistry. McGraw-Hill: Boston, 2002. 57. Peters, T., Serum Albumin. Advances in Protein Chemistry 1985, 37, 161-245. 58. Liu, J. Y.; Wang, Z. Y.; Liu, F. D.; Kane, A. B.; Hurt, R. H., Chemical Transformations of Nanosilver in Biological Environments. Acs Nano 2012, 6, (11), 9887-9899. 59. Maffre, P.; Nienhaus, K.; Amin, F.; Parak, W. J.; Nienhaus, G. U., Characterization of protein adsorption onto FePt nanoparticles using dual-focus fluorescence correlation spectroscopy. Beilstein Journal of Nanotechnology 2011, 2, 374-383. 60. Allouni, Z. E.; Cimpan, M. R.; Hol, P. J.; Skodvin, T.; Gjerdet, N. R., Agglomeration and sedimentation of TiO2 nanoparticles in cell culture medium. Colloid Surface B 2009, 68, (1), 83-87. 61. Dockal, M.; Carter, D. C.; Ruker, F., Conformational transitions of the three recombinant domains of human serum albumin depending on pH. Journal of Biological Chemistry 2000, 275, (5), 3042-3050. 62. Carter, D. C.; Ho, J. X., Structure of serum albumin. In Advances in Protein Chemistry; Lipoproteins, apolipoproteins, and lipases, Schumaker, V. N., Ed. 1994; Vol. 45, pp 153-203. 63. Era, S.; Sogami, M., H-1-NMR and CD studies on the structural transition of serum albumin in the acidic region - The N -> F transition. Journal of Peptide Research 1998, 52, (6), 431-442. 64. Zimmermann, R.; Kuttner, D.; Renner, L.; Kaufmann, M.; Zitzmann, J.; Muller, M.; Werner, C., Charging and structure of zwitterionic supported bilayer lipid membranes studied by streaming current measurements, fluorescence microscopy, and attenuated total reflection Fourier transform infrared spectroscopy. Biointerphases 2009, 4, (1), 1-6. 65. McCracken, C.; dutta, P.; Waldman, W. J., Critical assessment of toxicological effects of ingested nanoparticles. Environmental Science: Nano 2016, In Press. 66. Rosenthal, T. B., The effect of temperature on the pH of blood and plasma in vitro. Journal of Biological Chemistry 1948, 173, (1), 25-30. 67. Choi, J.; Reipa, V.; Hitchins, V. M.; Goering, P. L.; Malinauskas, R. A., Physicochemical Characterization and In Vitro Hemolysis Evaluation of Silver Nanoparticles. Toxicol Sci 2011, 123, (1), 133-143. 68. Docter, D.; Westmeier, D.; Markiewicz, M.; Stolte, S.; Knauer, S. K.; Stauber, R. H., The nanoparticle biomolecule corona: lessons learned - challenge accepted? Chemical Society reviews 2015, 44, (17), 6094-6121.

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(b) d∆F/dt  (Hz/min)

d∆F/dt  (Hz/min)

(a)

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0.1

0.01 100

0.1

0.01 1

1000

NaCl Concentration (mM)

10

CaCl2 Concentration (mM) Attachment Efficiency

Attachment Efficiency

580

(c) 1

0.1 10

100

1000

1

(d)

0.1

1

NaCl Concentration (mM)

10

CaCl2 Concentration (mM)

581 582

Figure 1. Deposition rates of citrate-coated AgNPs on silica surfaces as functions of (a) NaCl and

583

(b) CaCl2 concentrations at pH 7. Attachment efficiencies of citrate-coated AgNPs on silica

584

surfaces as functions of (c) NaCl and (d) CaCl2 concentrations at pH 7. The dashed lines in

585

Figure 1c and d are extrapolations through the favorable and unfavorable deposition regimes.

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4 DI 2 0 -2 -4 -6 -8 -10 -12 -14 -16 0

300 mM NaCl

AgNP deposition 300 mM 1 mM in 300 mM NaCl NaCl NaCl

(a) 50

100

150

200

Time (min)

Frequency Shift (Hz)

588

2 DI 1 0 -1 -2 -3 -4 -5 -6 -7 0

3 mM CaCl2

AgNP deposition       3mM 1mM 1mM in 3 mM CaCl2       CaCl2CaCl2 NaCl

(b) 50

100

150

200

Time (min) 589 590

Figure 2. Deposition and release of citrate-coated AgNPs on silica surfaces at pH 7. AgNPs

591

were deposited in the presence of (a) 300 mM NaCl and (b) 3 mM CaCl2.

592

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d∆F/dt (Hz/min)

1

0.1

(a)

0.01

10

100

Frequency Shift (Hz)

10 0

Formation of DOPC SLBs

300 mM AgNP deposition 300 mM NaCl in 300 mM NaCl NaCl

1 mM NaCl

-10 -20 -30 -40 -50 -60

(c)

-70 0

1000

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100

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NaCl Concentration (mM) 593

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1

0.1

0.01

(b) 10

100

Frequency Shift (Hz)

Attachment Efficiency

-25

1000

300 mM NaCl

-30

-35

-40

1mM NaCl

7.0 Hz Buffer effect

5.5 Hz AgNP deposition

3.6 Hz AgNP release

(d) 100

150

200

Time (min)

NaCl Concentration (mM) 594 595 596

Figure 3. (a) Deposition rates of citrate-coated AgNPs on DOPC SLBs as a function of NaCl

597

concentration at pH 7. (b) Attachment efficiencies of citrate-coated AgNPs on DOPC SLBs as a

598

function of NaCl concentration at pH 7. (c) Frequency shifts during the formation of a DOPC

599

SLB followed by the deposition (at 300 mM NaCl) and release of citrate-coated AgNPs on the

600

SLB at pH 7. (d) Close-up of the frequency shifts during the deposition and release of AgNPs on

601

the SLB at pH 7.

602 603

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AgNPs HSA-modified AgNPs

-0.6 -0.8 -1.0 -1.2 -1.4 -1.6

(a) 1

10

100

615 616 617

Electrophoretic Mobility (10-8m2/Vs)

1.5

614

AgNPs HSA-modified AgNPs

(c) 10

AgNPs HSA-modified AgNPs

1.0 0.5 0.0 -0.5 -1.0 -1.5 -2.0

(b) 1

10

100

200 180 160 140 120 100 80 60 40 0

30

40

50

AgNPs HSA-modified AgNPs

(d) 10

20

30

40

50

Time (min)

NaCl Concentration (mM)

618

20

Time (min)

612 613

200 180 160 140 120 100 80 60 40 0

NaCl Concentration (mM)

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Hydrodynamic Diameter (nm)

607

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Electrophoretic Mobility (10-8m2/Vs)

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619 620 621

Figure 4. EPMs of citrate-coated AgNPs (squares) and HSA-modified AgNPs (circles) as

622

functions of NaCl concentration at (a) pH 7 and (b) pH 2. Aggregation profiles of citrate-coated

623

AgNPs (squares) and HSA-modified AgNPs (circles) in the presence of 150 mM NaCl at (c) pH

624

7 and (d) pH 2.

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2 1

(a)

HSA

0 -1

HSA-modified AgNPs

-2 -3 -4

AgNPs

-5 -6 0

5

10

15

20

25

30

Time (min) 626

Frequency Shift (Hz)

2 1

(b) HSA

0 -1 -2

AgNPs

-3 -4 -5 -6 0

HSA-modified AgNPs

5

10

15

20

25

Time (min) 627 628

Figure 5. Frequency shifts during the deposition of HSA proteins (black), citrate-coated AgNPs

629

(blue), and HSA-modified AgNPs (red) on DOPC SLBs in the presence of 150 mM NaCl at (a)

630

pH 7 and (b) pH 2.

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pH 7

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HSA-modified AgNPs

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AgNPs 5

10

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25

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Time (min) 634 635 636 637 638

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