Influence of Solution Chemistry and Soft Protein Coronas on the

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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

1 2 3 4

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

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

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

ACS Paragon Plus Environment


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36

Abstract

37

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-

40

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

41

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

42

unmodified silica surfaces increased with increasing electrolyte concentrations under neutral pH

43

conditions.

44

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

45

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

46

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

47

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

48

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.

50

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

51

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

52

between AgNPs and SLBs.

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

This

53 54

Introduction

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

56

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

60

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

81

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

25

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

3



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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.

110 111 112

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).

119

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

134

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-

150

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

152

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

153

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

154

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

155

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

156

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



159

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

160

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

161

experimental condition.

162

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

163

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

164

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

169

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

170

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

171

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

174

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

179

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

180

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

184

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

48

To obtain the favorable deposition rates of AgNPs, the deposition

For the release experiments, the reversibility of AgNPs deposition was

6


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190 191

Results and Discussion

192

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

194

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

199

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

200 201

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.

203

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

207

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

208

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

210

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

217

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

219

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

220

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


These attachment

For both NaCl and CaCl2


221

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

222

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

223

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

224

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

228

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

238

(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

241

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

242

to low ionic strength solutions.

243

FIGURE 2

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

245

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

246

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

247

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

248

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

249

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

253

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

254

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

255

deposited AgNPs.

256

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

258

in Figure 3a.

259

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

260

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

262

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

263

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

264

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

265

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

266

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

267

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

268

mM.

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

269

FIGURE 3

270

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

271

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

272

coated crystals.

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

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

281

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

282

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

9


48

the


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283

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

284

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

285

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

286

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

287

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

288

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

289

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

290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

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

298

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

299

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

300

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

301

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

302

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

303

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

304

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

305

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

306

FIGURE 4

307

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

308

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

309

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

310

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

311

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

312

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

313

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


Page 11 of 23


314

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

315

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

316

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

317

concentrations due to charge screening effects.

318

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

319

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

320

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

321

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

322

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

323

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

324

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

325

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

326

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

327

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

328

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

329

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

330

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

331

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

332

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

333

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

334

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

335

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

11


HSA can undergo structural

Additionally, due to the lower


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.

Page 12 of 23

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

12


Page 13 of 23

376


Implications.

In order to investigate the toxicity of AgNPs in environmental and

377

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

405

Additional figures and details for Materials and Methods are presented.

406

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


This material is


Page 14 of 23

407

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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.

578 579

17



(b) d∆F/dt  (Hz/min)

d∆F/dt  (Hz/min)

(a)

Page 18 of 23

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


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.

586 587

18



Frequency Shift (Hz)

Page 19 of 23

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)


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

19



d∆F/dt (Hz/min)

1

0.1

(a)

0.01

10

100


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

Page 20 of 23

50

100

150

200

Time (min)

NaCl Concentration (mM) 593

AgNP deposition in 300 mM NaCl

1

0.1

0.01

(b) 10

100


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

20


608 609 610

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


(c) 10


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


(d) 10

20

30

40

50

Time (min)


618

20

Time (min)

612 613

200 180 160 140 120 100 80 60 40 0


611

Hydrodynamic Diameter (nm)

607

-0.4

Electrophoretic Mobility (10-8m2/Vs)

604 605 606


Hydrodynamic Diameter (nm)

Page 21 of 23

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.

625

21




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


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.

631

22


Page 22 of 23

Page 23 of 23

632


TOC Art

633


1 0

pH 7

-1

HSA-modified AgNPs

-2 -3 -4 0

AgNPs 5

10

15

20

25

30

Time (min) 634 635 636 637 638

23


Influence of Solution Chemistry and Soft Protein Coronas on the

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