Adsorption of Human Serum Albumin on Graphene Oxide


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Adsorption of Human Serum Albumin on Graphene Oxide: Implications for Protein Corona Formation and Conformation Xitong Liu, Chenxu Yan, and Kai Loon Chen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b03451 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 5, 2018

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Adsorption of Human Serum Albumin on Graphene Oxide: Implications for

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Protein Corona Formation and Conformation

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Revised: November 27, 2018

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Environmental Science & Technology

7 Xitong Liu,†,‡ Chenxu Yan,†,§ and Kai Loon Chen*,†,¶

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†Department

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of Environmental Health and Engineering, Johns Hopkins University, Baltimore, Maryland 21218-2686

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* Corresponding author: Kai Loon Chen, E-mail: [email protected], Phone: (202) 364-

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3128.

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Present addresses:

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‡Department

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Ave., Pittsburgh, Pennsylvania, 15213, United States

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§Department

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Austin, 301 East Dean Keeton Street, Stop C1786, Austin, Texas, 78712, United States

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¶DC

of Civil and Environmental Engineering, Carnegie Mellon University, 5000 Forbes

of Civil, Architectural and Environmental Engineering, University of Texas at

Water, 3900 Donaldson Place NW, Washington, DC 20016

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Abstract

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The influence of solution chemistry on the adsorption of human serum albumin (HSA)

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proteins on graphene oxide (GO) was investigated through batch adsorption experiments and the

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use of a quartz crystal microbalance with dissipation (QCM-D). The conformation of HSA layers

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on GO was also examined with the QCM-D. Our results show that an increase in ionic strength

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under neutral pH conditions resulted in stronger binding between HSA and GO, as well as more

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compact HSA layers on GO, emphasizing the key role of electrostatic interactions in controlling

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HSA-GO interactions. Calcium ions also facilitated HSA adsorption likely through charge

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neutralization and bridging effect.

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maximum HSA adsorption was observed at the isoelectric point of HSA (4.7). Under acidic

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conditions, the adsorption of HSA on GO led to the formation of protein layers with a high degree

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of fluidity due to the extended conformation of HSA. Finally, the attachment of GO to a supported

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lipid bilayer that was composed of zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine, a

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model for cell membranes, was reduced in the presence of protein coronas. This reduction in GO

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attachment was influenced by the conformation of the protein coronas on GO.

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Introduction

At physiological ionic strength conditions (150 mM),

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Owing to its unique optical,1 mechanical,2 chemical,3 and antibacterial properties,4

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graphene oxide (GO) has recently found widespread applications in diverse fields including energy

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storage,5 water treatment,6-8 and sensing of biomolecules.9 In particular, GO has been proposed as

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a promising candidate for efficient cellular imaging and drug delivery.10, 11 Recently, GO has been

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reported to show cyto- and geno-toxicity toward human cells.12, 13 Studies on the colloidal stability

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of GO nanomaterials14-17 and their transport in saturated porous media18 indicate that GO 2 ACS Paragon Plus Environment

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nanomaterials have high mobility in solution chemistries of natural surface waters. A mechanistic

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understanding of how GO nanosheets interact with biological systems, either when they are

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uptaken by organisms (e.g., from GO-contaminated water) or when they are intentionally

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administered into the human body (e.g., use of GO for drug delivery), will enable a better

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assessment of the biological impact of GO.

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When nanoparticles enter a biological fluid (such as blood plasma), proteins in the fluid

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can adsorb on the nanoparticle surface to form protein coronas.19-22 Previous work has shown that

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protein attachment leads to an increase in the colloidal stability of graphene nanomaterials.23, 24

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Moreover, protein coronas on nanoparticles are expected to have important implications for the

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biological impact of nanoparticles. Specifically, the coronas bound to nanoparticle surface confer

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a biological identity to the nanoparticles and may play critical roles in determining the cellular

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internalization and the distribution of nanoparticles when they enter a human body.19, 21, 25 In their

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study of the interactions of silica nanoparticles with A549 lung epithelial cells, Lesniak et al.26

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reported that weaker nanoparticle adhesion to cell membranes and lower internalization efficiency

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were observed when the nanoparticles were coated with protein coronas.

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The formation of protein coronas on GO has been the subject of several recent studies.

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Chong et al.27 demonstrated that the toxicity of GO toward A549 cells was decreased when the

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GO was coated with bovine serum albumin (BSA). They also observed an alteration in the

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secondary structures of BSA upon binding to GO.27 Lim and co-workers28 reported that the

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wavelength of the maximum fluorescence emission of human serum albumin (HSA) was red-

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shifted, which is indicative of conformational change of the proteins,29 upon adsorption on GO.

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Despite these findings, there is still a lack of understanding of the influence of solution chemistry,

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which will vary among different biological compartments,30 on the amount of proteins adsorbed 3 ACS Paragon Plus Environment

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on GO and the conformation of protein coronas. Moreover, our previous work has shown that GO

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can disrupt lipid membranes upon favorable attachment.31 It remains to be elucidated whether the

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presence of protein coronas will inhibit the attachment of GO to lipid membranes.

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In this study, we investigate the adsorption of HSA, which is the most abundant protein in

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human plasma,32 on GO. Adsorption isotherms of HSA on GO were obtained through batch

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experiments at different solution chemistries.

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information on the “dry” mass of adsorbed HSA on GO (i.e., mass of adsorbed proteins not

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including the mass of water associated with proteins).33 The adsorption of HSA on GO was also

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investigated through quartz crystal microbalance with dissipation (QCM-D) measurements by

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employing GO-coated sensors. The QCM-D allows for the determination of the “wet” mass of

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protein layers (i.e., mass of adsorbed proteins with water associated with proteins)33 on GO. In

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addition, the QCM-D measurements provide information on the initial kinetics of protein

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adsorption. The combination of batch and QCM-D experiments can thus enable a comprehensive

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understanding of the formation and conformation of protein coronas on GO. Finally, the impact

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of HSA adsorption on the attachment of GO to a supported lipid bilayer (SLB), which is used as a

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model for cell membranes,34 was investigated. Our findings provide fundamental insights into the

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interactions of HSA proteins with GO and will enable a better understanding of the biological

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impacts of GO in different biological fluids.

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Materials and Methods

Those isotherms will provide quantitative

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Preparation and Characterization of GO Suspensions. GO powder was purchased from

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Graphene Laboratories Inc. (Calverton, NY). According to the supplier, the GO contains 79%

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carbon and 20% oxygen. Atomic force microscopy (AFM) imaging was performed to measure the 4 ACS Paragon Plus Environment

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thickness of the GO nanosheets. Electrophoretic mobilities (EPMs) of the GO were also measured

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under varying solution chemistry conditions. Detailed procedures for the preparation of GO

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suspensions and AFM and EPM characterization are described in the Supporting Information (SI).

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Solution Chemistry. ACS-grade NaCl, CaCl2, and NaHCO3 were used for the preparation

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of stock solutions. All stock solutions were filtered through 0.1-m polyvinylidene fluoride filters

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(Millipore, MA) prior to use. The adsorption of HSA on GO was carried out under three pH

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conditions: pH 7.0  0.5, buffered with NaHCO3; pH 2.0  0.1, adjusted with 1 M HCl; and pH

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4.7  0.4, without pH adjustment.

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Batch Adsorption Experiments. Lyophilized HSA powder (≥ 97%, Sigma-Aldrich) was

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used for the preparation of HSA solutions. Adsorption of HSA on GO was carried out in low-

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protein binding centrifuge tubes (LoBind, Eppendorf) at room temperature (20°C).

The

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experiments were carried out in triplicates. After equilibrating the mixtures of HSA and GO for 6

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h, the GO with adsorbed HSA was separated from the mixture through centrifugation and the

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concentration of HSA in the supernatant was measured using a TOC Analyzer (TOC-L,

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Shimadzu). The amount of protein adsorbed on GO was calculated through material balance. We

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have performed adsorption kinetic studies at an initial HSA concentration of 125 mg/L and verified

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that the difference in HSA adsorption between 6 h and 8 h was less than 3%, indicating that the

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duration of adsorption of 6 h was sufficient for reaching adsorption equilibrium (SI Figures S1 and

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S2).35 Detailed procedures are presented in the SI.

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The isotherms of HSA on GO were fit to the Freundlich model as the assumptions for

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Langmuir isotherm model (e.g., homogeneous adsorption sites and no adsorbate-adsorbate

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interactions) have been reported in some studies to be invalid in the case of protein adsorption on

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solid surfaces.36, 37 The Freundlich model used to fit the isotherms is: 5 ACS Paragon Plus Environment

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q  K FCW n

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(1)

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where q (mg HSA/mg GO) and Cw (mg/L) are the equilibrium adsorbed and solution

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concentrations of HSA, respectively; KF [(mg HSA)1-n Ln (mg GO)-1] is the Freundlich affinity

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coefficient; and n (unitless) is the Freundlich linearity index.38 The parameter KF reflects the

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adsorption density and can be understood as indication of the overall binding strength between the

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adsorbate and adsorbent.39

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Adsorption of HSA Proteins on GO-Coated QCM-D Sensors. A QCM-D E4 setup

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(Biolin Scientific, Vas̈tra Frölunda, Sweden) was used for the investigation of HSA adsorption on

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GO surfaces under continuous-flow conditions. We first employed a layer-by-layer assembly

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technique to assemble GO layers on 5-MHz quartz crystal sensors with silica-coated surfaces

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(QSX 303). The sensor was first coated with positively charged poly-L-lysine (PLL). The PLL-

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modified sensor was then exposed to a GO suspension to allow for the formation of a GO layer on

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top of the PLL layer. We verified that the coverage of GO layers was complete (i.e., no exposed

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regions larger than 5  5 nm). Details are provided in the SI (Additional Materials and Methods

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and SI Figures S3 and S4). Following the coating of QCM-D sensors by GO, an electrolyte

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solution of interest was flowed through the chambers until a stable baseline was attained. A HSA

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solution (125 mg/L) prepared in the same electrolyte solution was introduced to the chambers to

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initiate the HSA adsorption process. After 2-h adsorption, the same electrolyte solution was

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flowed through the chambers to test the reversibility of HSA adsorption on GO. All QCM-D

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experiments were conducted in duplicates at 25°C under a flow rate of 0.1 mL/min, which results

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in laminar flow in the chambers.40

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The deposition or adsorption of mass on the sensors results in a decrease in the resonance

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frequency of the sensor (∆f), which can be related to the mass added on the sensor (∆m) according

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to the Sauerbrey equation:41

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m  C

f n

(2)

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where C is the mass sensitivity factor (= 17.7 ng cm-2 Hz-1 at 5 MHz) and n is the overtone number

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(1, 3, 5, 7, 11, 13). The Sauerbrey relation can be applied for sufficiently rigid and thin layers that

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are evenly distributed over the sensor surface.42 The adsorption of a viscoelastic layer will result

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in an enhancement in the ability of the sensor to dissipate energy, which is represented by the

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dissipation factor D.43 The ∆D/∆f values for HSA layers were calculated from the total frequency

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and dissipation shifts (in 7th overtone) resulting from the adsorption of HSA on GO. Under most

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conditions employed in HSA adsorption experiments, the ratio ∆D/∆f was lower than 0.12  10-6

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/Hz. Since the Sauerbrey equation is valid when the ratio of ∆D/∆f is significantly less than 0.4 

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10-6/Hz,44 the wet mass of HSA layer on GO was estimated using the Sauerbrey model.

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Deposition of GO and HSA-Modified GO on Model Cell Membranes. To examine the

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influence of HSA adsorption on the interactions of GO with model cell membranes, we

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investigated the rate of deposition of GO and HSA-modified GO on SLBs composed of

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zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) using the approach discussed in

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our earlier papers.31, 45-47 The HSA-modified GO was prepared by equilibrating a mixture of HSA

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and GO for 20 min to allow for the adsorption of HSA on GO. A GO or HSA-modified GO

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suspension prepared in the same solution chemistry was flowed across DOPC SLBs formed on

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silica-coated QCM-D sensors. The rate of deposition, expressed as the rate of frequency shift

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(Hz/min), was calculated by performing a linear regression during the initial 10 min of GO

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deposition. Details are presented in the SI. 7 ACS Paragon Plus Environment

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Results and Discussion

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Characterization of GO. AFM imaging revealed that the thickness of the GO nanosheets

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was ca. 1 nm (Figure 1a and b), consistent with the reported thickness of monolayer GO.48 The

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EPMs of GO were measured at 150 mM NaCl under varying pH conditions (Figure 1c). Over the

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pH range of 2.0–7.0, GO was negatively charged owing to the deprotonation of carboxyl groups

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on the edges and basal planes of GO.3 A decrease in pH from 7.0 to 2.0 resulted in a reduction in

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the magnitude of the EPM (Figure 1c), which is expected since fewer carboxyl groups were

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dissociated under lower pH conditions. At an ionic strength of 1 mM, the magnitude of the EPM

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was smaller in calcium than in sodium (Figure 1d). The presence of CaCl2 reduced the EPMs

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through charge neutralization stemming from the binding of calcium ions to the dissociated

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carboxyl groups.49

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FIGURE 1

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Influence of Ionic Strength on HSA Adsorption on GO. The adsorption of HSA on GO

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was investigated at 1, 30, and 150 mM NaCl at pH 7.0. The NaCl concentrations of 30 and 150

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mM are representative of the ionic strengths of human saliva and blood, respectively.50, 51 The use

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of 1 mM ionic strength, along with 30 and 150 mM, will provide insights into the role of

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electrostatic forces in controlling the adsorption of HSA on GO. The adsorption isotherms are

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presented in Figure 2a and the fitted parameters (KF and n) are shown in SI Table S1. The dry

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adsorbed HSA mass (or adsorption density, q) increased with increasing the equilibrium HSA

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concentration in solution (Cw) for all three NaCl concentrations, namely, 1, 30, and 150 mM. It is

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notable that KF increases with increasing NaCl concentration, suggesting that the overall binding

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strength between HSA and GO increases with the rise in ionic strength. The isoelectric point (IEP)

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of HSA has been reported as 4.7–5.8.52-54 At pH 7.0, therefore, both HSA and GO carry net 8 ACS Paragon Plus Environment

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negative charge. The overlapping of electrical double layers (EDLs) of the protein and the

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adsorbent is enthalpically unfavorable,37 resulting in a lower binding affinity between HSA and

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GO at 1 mM than at 30 or 150 mM.

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FIGURE 2

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The “wet” mass of protein layers adsorbed on GO at 1, 30, and 150 mM NaCl when

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exposed to HSA solutions with a concentration of 125 mg/L is presented in Figure 2b. This “wet”

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mass is calculated from the frequency shifts during the exposure of GO-coated QCM-D sensors to

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HSA (SI Figure S5) using the Sauerbrey model. For all three NaCl concentrations, the wet mass

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of adsorbed HSA layers increased with time over the course of 2 h. The HSA layers were

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subsequently rinsed with the same electrolyte solutions. Negligible release of HSA upon rinsing

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was observed for all three NaCl concentrations, indicating that the adsorption of HSA on GO was

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largely irreversible.

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Furthermore, Figure 2b shows that the wet mass of the HSA layers at 30 mM and 150 mM

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was larger than that at 1 mM, consistent with the trend for the dry mass of adsorbed HSA at the

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HSA concentration of 125 mg/L (Figure 2a). Interestingly, the wet mass at 150 mM was lower

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than 30 mM while the dry mass of adsorbed HSA at 150 mM was higher than 30 mM at the HSA

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concentration of 125 mg/L. This discrepancy between wet and dry masses indicates that, despite

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more HSA molecules adsorbed on GO at 150 mM than 30 mM, the HSA molecules were

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considerably more hydrated at 30 mM than 150 mM, resulting in the wet mass to be higher at 30

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mM.

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The conformation of the adsorbed HSA layer on GO under varying ionic strength

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conditions was investigated by comparing the ∆D/∆f values (Figure 2c). Higher ∆D/∆f values

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indicate that the adsorbed layer is more fluid or hydrated, whereas lower ∆D/∆f values are 9 ACS Paragon Plus Environment

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indicative of more rigid/compact layers.55, 56 The ∆D/∆f value for the HSA layer was highest at 1

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mM and lowest at 150 mM, indicating that the layer was most fluid at 1 mM and most rigid at 150

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mM (inset of Figure 2c). At 150 mM, the electrostatic repulsion between like-charged groups

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(such as carboxyl groups) within a single HSA molecule was reduced due to charge screening and

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the HSA molecules are thus expected to take more compact conformation.57, 58 In addition, the

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reduction in the intermolecular repulsion between adsorbed HSA molecules at 150 mM can lead

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to a denser distribution of HSA on GO surfaces and thus less water being trapped in the HSA

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layers. It is noteworthy that QCM-D does not directly provide information about the secondary

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structure of HSA. The use of other techniques, such as circular dichroism, will be required to

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unravel the change in the secondary structure of HSA upon adsorbing on GO.59

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The rates of HSA adsorption on GO were investigated by comparing the initial slopes of

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the mass-time plots at different ionic strengths (Figure 2b). The initial slope at 1 mM was much

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smaller than that at 30 and 150 mM, indicating that the slowest increase in wet mass occurred at 1

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mM. Since the adsorbed HSA layers were most hydrated at 1 mM (Figure 2c), this slow increase

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in wet mass also means that the slowest increase in HSA dry mass takes place at 1 mM, which is

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consistent with the adsorption kinetics obtained from batch experiments (SI Figure S1). The

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slowest HSA adsorption at 1 mM is attributed to the presence of an energy barrier to the adsorption

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of HSA due to strong EDL repulsions between HSA and GO.

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Influence of Calcium on HSA Adsorption on GO. Calcium ions are a key constituent in

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biological fluids and are present in human plasma at a concentration of ca. 1 mM.60, 61 The effect

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of calcium ions on the adsorption of HSA on GO at neutral pH and an ionic strength of 1 mM was

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investigated. The dry mass of adsorbed HSA on GO increased with increasing Cw both in the

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presence and absence of calcium (Figure 3a). In the presence of calcium ions (0.33 mM), the dry 10 ACS Paragon Plus Environment

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mass of adsorbed HSA and the fitted KF (SI Table S1) were both higher, indicating stronger

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binding between HSA and GO. Greater wet mass of adsorbed HSA layers was also observed in

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the presence of calcium (Figure 3b). The lower ∆D/∆f value in the presence of calcium (Figure

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3c) indicates that calcium results in a more compact HSA layer (inset in Figure 3c). The faster

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rate of wet mass uptake (greater initial slope in Figure 3b) and the higher degree of compactness

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of HSA layers (Figure 3c) suggest that the initial increase in the adsorbed dry mass of HSA was

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faster in the presence of calcium.

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FIGURE 3

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The presence of calcium ions results in a reduction in the magnitude of the EPMs of GO

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(Figure 1d). Furthermore, calcium ions can neutralize the charge of HSA molecules through

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binding to the imidazole and deprotonated carboxyl groups of HSA.62 Hence, the EDL repulsion

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between HSA and GO was reduced in the presence of calcium, resulting in faster initial adsorption

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and higher dry mass adsorption. The binding of calcium ions to the carboxyl groups may also lead

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to the bridging of amino acids in HSA63 and hence the compaction of individual HSA molecules.

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Therefore, a rigid/compact HSA layer was formed on GO in the presence of calcium.

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Influence of pH on HSA Adsorption on GO. The adsorption of HSA on GO was

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investigated at pH 2.0, 4.7, and 7.0, which are lower than, equal to, and higher than the IEP of

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HSA, respectively. These pH conditions are also close to the pH of several biological fluids. For

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example, the pH values of human gastric fluids, duodenal fluids, and blood are ca. 1–3, ca. 5–6,

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and ca. 7.4, respectively.64-66 The ionic strength for these experiments was 150 mM, which is the

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ionic strength of most biological fluids.21 Figure 4a shows that the dry mass of adsorbed HSA

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increased with increasing Cw for all three pH conditions. At the HSA concentration of 125 mg/L,

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the dry mass of adsorption was considerably higher at pH 4.7 than 2.0 and 7.0. The wet mass of 11 ACS Paragon Plus Environment

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HSA layer adsorbed on GO at the same HSA concentration of 125 mg/L (Figure 4b) however

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followed the order pH 2.0 > pH 4.7 > pH 7.0. It is noteworthy that, at pH 2.0, the ∆D/∆f value

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was high (ca. 0.5  10-6/Hz), and thus the wet mass estimated using the Sauerbrey model is

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underestimated under this condition.33, 67 The discrepancy in trends for the dry and wet masses,

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particularly at pH 2.0 and 4.7, suggests that more water was trapped in the adsorbed HSA layer at

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pH 2.0 than at 4.7. This proposition is further supported by the observation that the ∆D/∆f value

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at pH 2.0 was the highest among all the three pH conditions and was over five times higher than

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that at pH 4.7 (Figure 4c). In contrast, the lowest ∆D/∆f value was observed at pH 4.7, implying

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that the adsorbed HSA layer took the most compact conformation at this pH condition (inset of

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Figure 4c).

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FIGURE 4

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The stronger adsorption and more compact HSA layer at pH 4.7 than at 7.0 (Figure 4a and

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c) is most likely due to the difference in the net charge of HSA under these two conditions.68 With

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the decrease in pH from 7.0 to 4.7, the surface of GO became less negatively charged (Figure 1c),

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and the net charge of HSA changed from negative to zero, thus eliminating the electrostatic

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repulsion between HSA and the GO layer and resulting in significant adsorption. At the IEP of

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HSA (i.e., pH 4.7), the protein molecules take the most compact structure as the intramolecular

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electrostatic repulsion is minimized.69 In addition, the adsorbed HSA molecules experience

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negligible intermolecular electrostatic repulsion. Both mechanisms allow for a dense distribution

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on GO surface at pH 4.7. Maximum adsorption near the IEP of HSA has also been reported for

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polystyrene,70 silicon,53 and TiO268 surfaces.

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When the pH is further lowered to 2.0, HSA carries a net positive charge. Despite the

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electrostatic attraction between HSA and GO, the lower dry mass of adsorbed HSA at pH 2.0 than 12 ACS Paragon Plus Environment

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pH 4.7 (Figure 4a) is likely due to the intermolecular repulsion between adsorbed HSA molecules

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and hence a more sparse distribution of HSA on GO. At pH 2.0, the electrostatic repulsion between

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positively charged amino acid residues within HSA leads to a structural transition to a fully

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extended conformation with a high aspect ratio.71-73 Under the elongated conformation at pH 2.0,

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the adsorbed HSA on GO may either take side-on (i.e., lying flat) or end-on (i.e., extending into

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the bulk solution) orientations. The pronounced dissipation shift caused by HSA adsorption (SI

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Figure S5c) and the high degree of the fluidity of HSA layers (Figure 4c) at pH 2.0 suggest that a

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considerable fraction of adsorbed HSA molecules take the end-on orientation, which allows for

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more residues in HSA being exposed to the bulk solution and associated with water molecules.74

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HSA Coronas Reduced GO Attachment to Model Cell Membranes. The influence of

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HSA coronas on the deposition of GO on SLBs was investigated at neutral pH conditions. In the

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absence of HSA, the rate of GO deposition on SLBs were similar at 30 and 150 mM NaCl (Figure

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5), which is consistent with our previous work which demonstrated that fast deposition of GO on

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DOPC SLBs was achieved at NaCl concentrations higher than ca. 30 mM.31 In the presence of

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HSA, the rates of deposition were reduced under both ionic strength conditions, but the extent of

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reduction in deposition was considerably greater at 30 mM than at 150 mM. This observation

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implies that the HSA coronas on GO had a more substantial impact on the interactions between

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GO and SLBs at 30 mM NaCl.

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FIGURE 5

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At both 30 and 150 mM NaCl, the average hydrodynamic size of GO was similar in the

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absence and presence of HSA over the course of the deposition experiments (SI Figure S6a). At

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the same time, the magnitude of the EPMs of GO did not change significantly in the presence of

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HSA under both conditions (SI Figure S6b). Therefore, the inhibition of GO deposition by HSA 13 ACS Paragon Plus Environment

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coronas at 30 and 150 mM NaCl was unlikely to result from a change in the aggregation behavior

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of GO or a rise in the negative surface charge of GO. As shown in Figure 2c, the HSA layer on

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GO at 30 mM was more fluid (or hydrated) compared to that at 150 mM NaCl. In their study on

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the deposition of humic matter-coated colloids on quartz sand, Amirbahman and Olson75,

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postulated that the magnitude of steric repulsion between the colloids and the sand was directly

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dependent upon the conformation of the adsorbed polymers. A more hydrated HSA corona is

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expected to impart a greater steric repulsion between HSA-coated GO and SLBs, thereby

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inhibiting the deposition of GO on SLBs more significantly.

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Implications. In order to better understand the formation and conformation of protein

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coronas on GO, the adsorption of HSA on GO was investigated under biologically relevant

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conditions. Our results show that the ionic strength and pH conditions, as well as the presence of

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calcium ions in the media, had key influences on both the HSA-GO binding strength and the

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conformation of the coronas on GO. Higher ionic strength (e.g., blood compared to saliva)51 can

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lead to enhanced binding of HSA to GO and more compact protein coronas on GO. Under acidic

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conditions (such as in gastric fluids), the protein coronas on GO can be highly hydrated due to the

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conformational changes of the proteins. These findings will provide fundamental insights into the

310

dynamic changes of protein coronas when GO is transported from one biological compartment to

311

another. We also show that the attachment of GO to lipid bilayers was reduced upon being coated

312

by protein coronas, with a higher degree of reduction observed with more fluid (hydrated) protein

313

coronas. Therefore, the hemolytic activity of GO12 may be mitigated by protein coronas to varying

314

degrees depending upon the conformation of the coronas. The formation of macromolecular

315

coatings on GO surface should be taken into account during the assessment of the cytotoxicity of

316

GO. 14 ACS Paragon Plus Environment

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317

Given the complexity of biological media, the coronas formed on nanoparticle surfaces

318

often include multiple components (e.g., fatty acids, lipids, and proteins)77 and undergo dynamic

319

nanoparticle-biomolecule association and dissociation.21 For example, a variety of proteins are

320

present in biological fluids at different concentrations and exhibit varying affinities to

321

nanoparticles.19 The association of other proteins with GO will lead to the dissociation of adsorbed

322

HSA and a change in the composition of the protein coronas on GO.78 Such an evolution of corona

323

composition can impact the uptake of nanoparticles by cells,79 which deserves further study.

324

Further investigations that involve the use of more realistic biological media are warranted to allow

325

for a holistic understanding of the biological impacts of GO.

326 327

Supporting Information

328

The Supporting Information is available free of charge on the ACS Publication website.

329

Additional materials and methods, fitted parameters of adsorption isotherms (Table S1),

330

adsorption kinetics (Figure S1), fitted parameters of adsorption kinetics (Table S2),

331

adsorption of HSA on GO at 6 and 8 h (Figure S2), TEM images of gold nanoparticles

332

(Figure S3), coating of a QCM-D sensor with a GO layer (Figure S4), frequency and

333

dissipation shifts during HSA adsorption on a GO layer (Figure S5), hydrodynamic

334

diameters and EPMs of GO (Figure S6).

335 336

Corresponding Author

337

*E-mail: [email protected] Phone: (202) 364-3128.

338 339

Notes 15 ACS Paragon Plus Environment

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340

The authors declare no competing financial interest.

341 342

Acknowledgements

343

This work was supported by the National Science Foundation (CBET-1605815). We thank

344

Michael McCaffery from the Integrated Imaging Center at Johns Hopkins University for

345

performing the TEM imaging.

346 347

References

348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377

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554 555

Profile 1 Profile 2 Profile 3

1.0 (b) 0.5

Z (nm)

556 557 558

0.0 -0.5 0

559

100

200

300

X (nm)

560 0.0

563 564 565 566 567

Electrophoretic Mobility -8 2 (10 m /Vs)

562

0.0

(c)

-0.5 -1.0 -1.5 -2.0 -2.5 1

2

3

4

5

6

7

8

Electrophoretic Mobility -8 2 (10 m /Vs)

561

-0.5 -1.0 -1.5 -2.0 -2.5 -3.0

(d) 1 mM NaCl

0.33 mM CaCl2

pH

568 569

Figure 1. (a) AFM image of GO. (b) AFM height profiles of GO. The positions of the profiles are

570

marked as white lines in (a). (c) EPMs of GO at 150 mM NaCl under different pH conditions. (d)

571

EPMs of GO at pH 7.0 in the absence and presence of calcium ions (ionic strength = 1 mM). Error

572

bars in (c) and (d) represent standard deviations.

573

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574 575 0.7

579

0.5 0.4 0.3 0.2 0.1

580 581 582

0.0

0

50

100

150

Cw (mg/L)

S+HSA

200

250

S

(c)

30 mM

600

Mass (ng/cm2)

578

0.6

q (mg/mg GO)

577

700

150 mM NaCl, pH 7.0 30 mM NaCl, pH 7.0 1 mM NaCl, pH 7.0

S

150 mM

500

D/f(7) (10-6)

(b)

576 (a)

400 1 mM

300 200 100 0 0

40

80

120

160

-0.16 -0.14 -0.12 -0.10 -0.08 -0.06 -0.04 -0.02 0.00

1 mM

30 mM

150 mM

NaCl Concentration

Time (min) 12 132

583

Figure 2. (a) Adsorption isotherms of HSA on GO at pH 7.0. Dashed lines represent Freundlich

584

fitting. (b) Wet mass of HSA adsorbed on a GO layer estimated using Sauerbrey model during the

585

exposure of the GO layer to 125 mg/L HSA at pH 7.0 followed by rinsing with background

586

electrolytes. Vertical lines indicate the time points at which solutions were switched from protein-

587

free solutions (S) to protein solutions (S+HSA) and finally back to protein-free solutions (S). (c)

588

Overall D/f values calculated from data in (b). The cartoon illustrates the proposed

589

conformation of HSA layers on GO under different ionic strengths.

590 591

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592

594

596 597 598

0.6

q (mg/mg GO)

595

0.33 mM CaCl2, pH 7.0 1 mM NaCl, pH 7.0

(b)

0.5 0.4 0.3 0.2 0.1 0.0

600

S

500

S+HSA

(c)

S

-0.14

0.33 mM CaCl2

-0.12

D/f(7) (10-6)

(a)0.7

Mass (ng/cm2)

593

400 300 1 mM NaCl

200 100

50

100

150

Cw (mg/L)

200

250

0

-0.08 -0.06 -0.04

132

-0.02

0 0

-0.10

40

80

120

160

0.00

1 mM NaCl 0.33 mM CaCl2

Time (min)

599 12

600

Figure 3. (a) Adsorption isotherms of HSA on GO at pH 7.0 in the absence and presence of

601

calcium ions. Dashed lines represent Freundlich fitting. Data for 1 mM NaCl is reproduced from

602

Figure 2a. (b) Wet mass of HSA adsorbed on a GO layer estimated using Sauerbrey model during

603

the exposure of the GO layer to 125 mg/L HSA at pH 7.0 followed by rinsing with background

604

electrolytes. Data for 1 mM NaCl is reproduced from Figure 2b. (c) Overall D/f values

605

calculated from data in (b). The cartoon illustrates the proposed conformation of HSA layers on

606

GO in absence and presence of calcium.

607

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611 612

0.8 0.6 0.4 pH 4.7, 150 mM NaCl pH 2.0, 150 mM NaCl pH 7.0, 150 mM NaCl

0.2 0.0

0

50

613

100

150

Cw (mg/L)

200

1000

S

800

S+HSA

(c)

S

-0.7

pH 2.0

600 400

pH 7.0

200 0

250

0

40

134

-0.6

pH 4.7

D/f(7) (10-6)

610

(b) Mass (ng/cm2)

609

q (mg/mg GO)

608 (a) 1.0

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80

120

Time (min)

160

-0.5 -0.4

12

-0.1

0.0

2.0

4.7

7.0

pH

614 615 616

Figure 4. (a) Adsorption isotherms of HSA on GO at 150 mM NaCl under varying pH conditions.

617

Dashed lines represent Freundlich fitting. Data for pH 7.0 is reproduced from Figure 2a. (b) Wet

618

mass of HSA adsorbed on a GO layer estimated using Sauerbrey model during the exposure of the

619

GO layer to 125 mg/L HSA at 150 mM NaCl followed by rinsing with background electrolytes.

620

Data for pH 7.0 is reproduced from Figure 2b. (c) Overall D/f values calculated from data in

621

(b). The cartoon illustrates the proposed conformation of HSA layers on GO under different pH

622

conditions.

623

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624

625

627 628 629 630 631 632

Deposition Rate (Hz/min)

626

1.0 0.8 0.6 0.4 0.2 0.0

30 mM

150 mM

NaCl Concentration

633 634

Figure 5. Effect of HSA adsorption on the deposition of GO on DOPC supported lipid bilayers at

635

pH 7.0 under different ionic strengths. Open bars: GO; solid bars: HSA-modified GO. The cartoon

636

illustrates the interactions of HSA-coated GO with lipid bilayers at 30 and 150 mM NaCl.

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637

TOC Art

638

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