Adsorption of Human Serum Albumin on Graphene Oxide

Subscriber access provided by TULANE UNIVERSITY

Environmental Processes

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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

Page 1 of 26

Environmental Science & Technology

1 2

Adsorption of Human Serum Albumin on Graphene Oxide: Implications for

3

Protein Corona Formation and Conformation

4 5

Revised: November 27, 2018

6


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

8 9

†Department

10 11

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

12 13

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

14

3128.

15 16

Present addresses:

17

‡Department

18

Ave., Pittsburgh, Pennsylvania, 15213, United States

19

§Department

20

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

21

¶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

1 ACS Paragon Plus Environment


22

Abstract

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

neutralization and bridging effect.

31

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

32

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

33

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

34

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

35

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

36

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

37

Introduction

At physiological ionic strength conditions (150 mM),

38

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

39

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

40

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

41

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

42

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

43

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

Page 2 of 26

Page 3 of 26


44

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

45

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

46

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

47

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

48

assessment of the biological impact of GO.

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

were observed when the nanoparticles were coated with protein coronas.

59

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

60

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

61

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

62

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

63

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

64

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

65

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

66

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


67

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

68

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

69

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

70

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

71

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

72

experiments at different solution chemistries.

73

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

74

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

75

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

76

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

77

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

78

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

79

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

80

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

81

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

82

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

83

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

84

impacts of GO in different biological fluids.

85

Materials and Methods

Those isotherms will provide quantitative

86

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

87

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

88

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

Page 4 of 26

Page 5 of 26


89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

4.7  0.4, without pH adjustment.

97

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

98

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

99

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

The

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

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

108

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

109

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

110

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

111

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


q  K FCW n

112

Page 6 of 26

(1)

113

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

114

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

115

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

116

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

117

adsorbate and adsorbent.39

118

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

119

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

120

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

121

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

122

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

123

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

124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

in laminar flow in the chambers.40


Page 7 of 26


133

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

134

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

135

to the Sauerbrey equation:41

136

m  C

f n

(2)

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

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

154

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

155

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


156

Results and Discussion

157

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

158

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

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

carboxyl groups.49

167

FIGURE 1

168

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

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

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

178

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

Page 8 of 26

Page 9 of 26


179

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

180

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

181

GO at 1 mM than at 30 or 150 mM.

182

FIGURE 2

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

largely irreversible.

191

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

192

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

193

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

194

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

195

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

196

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

197

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

198

mM.

199

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

200

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

201

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


202

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

203

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

204

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

205

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

206

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

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

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

217

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

218

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

219

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

220

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

221

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

222

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

223

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

224

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

Page 10 of 26

Page 11 of 26


225

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

226

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

227

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

228

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

229

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

230

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

231

faster in the presence of calcium.

232

FIGURE 3

233

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

234

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

235

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

236

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

237

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

238

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

239

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

240

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

241

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

242

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

243

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

244

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

245

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

246

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

247

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


248

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

249

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

250

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

251

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

252

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

253

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

254

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

255

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

256

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

257

Figure 4c).

258

FIGURE 4

259

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

260

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

261

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

262

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

263

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

264

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

265

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

266

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

267

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

268

polystyrene,70 silicon,53 and TiO268 surfaces.

269

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

270

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

Page 12 of 26

Page 13 of 26


271

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

272

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

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

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

281

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

282

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

283

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

284

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

285

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

286

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

287

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

288

GO and SLBs at 30 mM NaCl.

289

FIGURE 5

290

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

291

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

292

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

293

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


Page 14 of 26

294

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

295

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

296

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

297

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

298

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

299

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

300

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

301

inhibiting the deposition of GO on SLBs more significantly.

76

302

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

303

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

304

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

305

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

306

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

307

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

308

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

309

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

Page 15 of 26


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


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

1. Loh, K. P.; Bao, Q. L.; Eda, G.; Chhowalla, M. Graphene oxide as a chemically tunable platform for optical applications. Nature Chem. 2010, 2, (12), 1015-1024. 2. Dikin, D. A.; Stankovich, S.; Zimney, E. J.; Piner, R. D.; Dommett, G. H. B.; Evmenenko, G.; Nguyen, S. T.; Ruoff, R. S. Preparation and characterization of graphene oxide paper. Nature 2007, 448, (7152), 457-460. 3. Dreyer, D. R.; Park, S.; Bielawski, C. W.; Ruoff, R. S. The chemistry of graphene oxide. Chem. Soc. Rev. 2010, 39, (1), 228-40. 4. Liu, S. B.; Zeng, T. H.; Hofmann, M.; Burcombe, E.; Wei, J.; Jiang, R. R.; Kong, J.; Chen, Y. Antibacterial Activity of Graphite, Graphite Oxide, Graphene Oxide, and Reduced Graphene Oxide: Membrane and Oxidative Stress. ACS Nano 2011, 5, (9), 6971-6980. 5. Xu, J. J.; Wang, K.; Zu, S. Z.; Han, B. H.; Wei, Z. X. Hierarchical Nanocomposites of Polyaniline Nanowire Arrays on Graphene Oxide Sheets with Synergistic Effect for Energy Storage. ACS Nano 2010, 4, (9), 5019-5026. 6. Perreault, F.; Fonseca de Faria, A.; Elimelech, M. Environmental applications of graphenebased nanomaterials. Chem. Soc. Rev. 2015, 44, (16), 5861-96. 7. Jiang, Y.; Wang, W. N.; Liu, D.; Nie, Y.; Li, W. L.; Wu, J. W.; Zhang, F. Z.; Biswas, P.; Fortner, J. D. Engineered Crumpled Graphene Oxide Nanocomposite Membrane Assemblies for Advanced Water Treatment Processes. Environ. Sci. Technol. 2015, 49, (11), 6846-6854. 8. Hu, M.; Mi, B. X. Enabling Graphene Oxide Nanosheets as Water Separation Membranes. Environ. Sci. Technol. 2013, 47, (8), 3715-3723. 9. Dong, H. F.; Gao, W. C.; Yan, F.; Ji, H. X.; Ju, H. X. Fluorescence Resonance Energy Transfer between Quantum Dots and Graphene Oxide for Sensing Biomolecules. Anal. Chem. 2010, 82, (13), 5511-5517. 10. Sun, X. M.; Liu, Z.; Welsher, K.; Robinson, J. T.; Goodwin, A.; Zaric, S.; Dai, H. J. NanoGraphene Oxide for Cellular Imaging and Drug Delivery. Nano Res 2008, 1, (3), 203-212. 11. Liu, Z.; Robinson, J. T.; Sun, X. M.; Dai, H. J. PEGylated nano-graphene oxide for delivery of water-insoluble cancer drugs. J. Am. Chem. Soc. 2008, 130, (33), 10876-10877. 12. Liao, K. H.; Lin, Y. S.; Macosko, C. W.; Haynes, C. L. Cytotoxicity of Graphene Oxide and Graphene in Human Erythrocytes and Skin Fibroblasts. ACS Appl. Mater. Interfaces 2011, 3, (7), 2607-2615. 16 ACS Paragon Plus Environment

Page 16 of 26

Page 17 of 26

378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422


13. Akhavan, O.; Ghaderi, E.; Akhavan, A. Size-dependent genotoxicity of graphene nanoplatelets in human stem cells. Biomaterials 2012, 33, (32), 8017-8025. 14. Chowdhury, I.; Duch, M. C.; Mansukhani, N. D.; Hersam, M. C.; Bouchard, D. Colloidal properties and stability of graphene oxide nanomaterials in the aquatic environment. Environ. Sci. Technol. 2013, 47, (12), 6288-96. 15. Wu, L.; Liu, L.; Gao, B.; Munoz-Carpena, R.; Zhang, M.; Chen, H.; Zhou, Z. H.; Wang, H. Aggregation Kinetics of Graphene Oxides in Aqueous Solutions: Experiments, Mechanisms, and Modeling. Langmuir 2013, 29, (49), 15174-15181. 16. Chowdhury, I.; Mansukhani, N. D.; Guiney, L. M.; Hersam, M. C.; Bouchard, D. Aggregation and Stability of Reduced Graphene Oxide: Complex Roles of Divalent Cations, pH, and Natural Organic Matter. Environ. Sci. Technol. 2015, 49, (18), 10886-10893. 17. Gudarzi, M. M. Colloidal Stability of Graphene Oxide: Aggregation in Two Dimensions. Langmuir 2016, 32, (20), 5058-5068. 18. Lanphere, J. D.; Luth, C. J.; Walker, S. L. Effects of Solution Chemistry on the Transport of Graphene Oxide in Saturated Porous Media. Environ. Sci. Technol. 2013, 47, (9), 4255-4261. 19. Lynch, I.; Dawson, K. A. Protein-nanoparticle interactions. Nano Today 2008, 3, (1-2), 4047. 20. Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K. A. Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, (38), 14265-14270. 21. Nel, A. E.; Madler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Understanding biophysicochemical interactions at the nano-bio interface. Nat. Mater. 2009, 8, (7), 543-557. 22. Huang, R. X.; Carney, R. R.; Ikuma, K.; Stellacci, F.; Lau, B. L. T. Effects of Surface Compositional and Structural Heterogeneity on Nanoparticle-Protein Interactions: Different Protein Configurations. ACS Nano 2014, 8, (6), 5402-5412. 23. Castagnola, V.; Zhao, W.; Boselli, L.; Lo Giudice, M. C.; Meder, F.; Polo, E.; Paton, K. R.; Backes, C.; Coleman, J. N.; Dawson, K. A. Biological recognition of graphene nanoflakes. Nature Communications 2018, 9. 24. Shen, J.; Shi, M.; Yan, B.; Ma, H.; Li, N.; Hu, Y.; Ye, M. Covalent attaching protein to graphene oxide via diimide-activated amidation. Colloid Surface B 2010, 81, (2), 434-438. 25. Monopoli, M. P.; Aberg, C.; Salvati, A.; Dawson, K. A. Biomolecular coronas provide the biological identity of nanosized materials. Nat. Nanotechnol. 2012, 7, (12), 779-786. 26. Lesniak, A.; Fenaroli, F.; Monopoli, M. R.; Aberg, C.; Dawson, K. A.; Salvati, A. Effects of the Presence or Absence of a Protein Corona on Silica Nanoparticle Uptake and Impact on Cells. ACS Nano 2012, 6, (7), 5845-5857. 27. Chong, Y.; Ge, C. C.; Yang, Z. X.; Garate, J. A.; Gu, Z. L.; Weber, J. K.; Liu, J. J.; Zhou, R. H. Reduced Cytotoxicity of Graphene Nanosheets Mediated by Blood-Protein Coating. ACS Nano 2015, 9, (6), 5713-5724. 28. Kenry; Loh, K. P.; Lim, C. T. Molecular interactions of graphene oxide with human blood plasma proteins. Nanoscale 2016, 8, (17), 9425-9441. 29. Aguanno, J. J.; Ladenson, J. H. Influence of Fatty-Acids on the Binding of Calcium to Human-Albumin - Correlation of Binding and Conformation Studies and Evidence for Distinct Differences between Unsaturated Fatty-Acids and Saturated Fatty-Acids. J. Biol. Chem. 1982, 257, (15), 8745-8748. 17 ACS Paragon Plus Environment


423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467

30. Marques, M. R. C.; Loebenberg, R.; Almukainzi, M. Simulated Biological Fluids with Possible Application in Dissolution Testing. Dissolution Technologies 2011, 18, (3), 15-28. 31. Liu, X.; Chen, K. L. Interactions of Graphene Oxide with Model Cell Membranes: Probing Nanoparticle Attachment and Lipid Bilayer Disruption. Langmuir 2015, 31, (44), 12076-86. 32. Rixman, M. A.; Dean, D.; Macias, C. E.; Ortiz, C. Nanoscale intermolecular interactions between human serum albumin and alkanethiol self-assembled monolayers. Langmuir 2003, 19, (15), 6202-6218. 33. Hook, F.; Voros, J.; Rodahl, M.; Kurrat, R.; Boni, P.; Ramsden, J. J.; Textor, M.; Spencer, N. D.; Tengvall, P.; Gold, J.; Kasemo, B. A comparative study of protein adsorption on titanium oxide surfaces using in situ ellipsometry, optical waveguide lightmode spectroscopy, and quartz crystal microbalance/dissipation. Colloid Surface B 2002, 24, (2), 155-170. 34. Chen, K. L.; Bothun, G. D. Nanoparticles Meet Cell Membranes: Probing Nonspecific Interactions. using Model Membranes. Environ. Sci. Technol. 2014, 48, (2), 873-880. 35. Ji, L.; Liu, F.; Xu, Z.; Zheng, S.; Zhu, D. Adsorption of Pharmaceutical Antibiotics on Template-Synthesized Ordered Micro- and Mesoporous Carbons. Environ. Sci. Technol. 2010, 44, (8), 3116-3122. 36. Latour, R. A. The Langmuir isotherm: A commonly applied but misleading approach for the analysis of protein adsorption behavior. J Biomed Mater Res A 2015, 103, (3), 949-958. 37. Norde, W. Adsorption of Proteins from Solution at the Solid-Liquid Interface. Adv. Colloid Interface Sci. 1986, 25, (4), 267-340. 38. Zhu, D. Q.; Pignatello, J. J. Characterization of aromatic compound sorptive interactions with black carbon (charcoal) assisted by graphite as a model. Environ. Sci. Technol. 2005, 39, (7), 2033-2041. 39. Benjamin, M. M.; Lawler, D. F., Water quality engineering: physical/chemical treatment processes. John Wiley & Sons: 2013. 40. Qu, X. L.; Alvarez, P. J. J.; Li, Q. L. Impact of Sunlight and Humic Acid on the Deposition Kinetics of Aqueous Fullerene Nanoparticles (nC60). Environ. Sci. Technol. 2012, 46, (24), 1345513462. 41. Sauerbrey, G. Verwendung Von Schwingquarzen Zur Wagung Dunner Schichten Und Zur Mikrowagung. Z Phys 1959, 155, (2), 206-222. 42. Hook, F.; Rodahl, M.; Kasemo, B.; Brzezinski, P. Structural changes in hemoglobin during adsorption to solid surfaces: Effects of pH, ionic strength, and ligand binding. Proc. Natl. Acad. Sci. U. S. A. 1998, 95, (21), 12271-12276. 43. Rodahl, M.; Hook, F.; Krozer, A.; Brzezinski, P.; Kasemo, B. Quartz-Crystal Microbalance Setup for Frequency and Q-Factor Measurements in Gaseous and Liquid Environments. Rev. Sci. Instrum. 1995, 66, (7), 3924-3930. 44. Reviakine, I.; Johannsmann, D.; Richter, R. P. Hearing What You Cannot See and Visualizing What You Hear: Interpreting Quartz Crystal Microbalance Data from Solvated Interfaces. Anal. Chem. 2011, 83, (23), 8838-8848. 45. Yi, P.; Chen, K. L. Interaction of Multiwalled Carbon Nanotubes with Supported Lipid Bilayers and Vesicles as Model Biological Membranes. Environ. Sci. Technol. 2013, 47, (11), 5711-5719. 46. Wang, Q. Y.; Lim, M. H.; Liu, X. T.; Wang, Z. W.; Chen, K. L. Influence of Solution Chemistry and Soft Protein Coronas on the Interactions of Silver Nanoparticles with Model Biological Membranes. Environ. Sci. Technol. 2016, 50, (5), 2301-2309. 18 ACS Paragon Plus Environment

Page 18 of 26

Page 19 of 26

468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512


47. Liu, X. T.; Chen, K. L. Aggregation and interactions of chemical mechanical planarization nanoparticles with model biological membranes: role of phosphate adsorption. Environ-Sci Nano 2016, 3, (1), 146-156. 48. Stankovich, S.; Dikin, D. A.; Piner, R. D.; Kohlhaas, K. A.; Kleinhammes, A.; Jia, Y.; Wu, Y.; Nguyen, S. T.; Ruoff, R. S. Synthesis of graphene-based nanosheets via chemical reduction of exfoliated graphite oxide. Carbon 2007, 45, (7), 1558-1565. 49. Yi, P.; Chen, K. L. Influence of surface oxidation on the aggregation and deposition kinetics of multiwalled carbon nanotubes in monovalent and divalent electrolytes. Langmuir 2011, 27, (7), 3588-99. 50. Dawes, C. Circadian-rhythms in human salivary flow-rate and composition. Journal of Physiology-London 1972, 220, (3), 529-545. 51. Macakova, L.; Yakubov, G. E.; Plunkett, M. A.; Stokes, J. R. Influence of ionic strength on the tribological properties of pre-adsorbed salivary films. Tribol Int 2011, 44, (9), 956-962. 52. Gianazza, E.; Frigerio, A.; Astruatestori, S.; Righetti, P. G. The Behavior of SerumAlbumin Upon Isoelectric-Focusing on Immobilized pH Gradients. Electrophoresis 1984, 5, (5), 310-312. 53. Ortega-Vinuesa, J. L.; Tengvall, P.; Lundstrom, I. Molecular packing of HSA, IgG, and fibrinogen adsorbed on silicon by AFM imaging. Thin Solid Films 1998, 324, (1-2), 257-273. 54. Langer, K.; Balthasar, S.; Vogel, V.; Dinauer, N.; von Briesen, H.; Schubert, D. Optimization of the preparation process for human serum albumin (HSA) nanoparticles. Int. J. Pharm. 2003, 257, (1-2), 169-180. 55. de Kerchove, A. J.; Elimelech, M. Structural growth and viscoelastic properties of adsorbed alginate layers in monovalent and divalent salts. Macromolecules 2006, 39, (19), 6558-6564. 56. Nguyen, T. H.; Elimelech, M. Plasmid DNA adsorption on silica: Kinetics and conformational changes in monovalent and divalent salts. Biomacromolecules 2007, 8, (1), 24-32. 57. Giancola, C.; DeSena, C.; Fessas, D.; Graziano, G.; Barone, G. DSC studies on bovine serum albumin denaturation - Effects of ionic strength and SDS concentration. Int. J. Biol. Macromol. 1997, 20, (3), 193-204. 58. Yamasaki, M.; Yano, H.; Aoki, K. Differential Scanning Calorimetric Studies on Bovine Serum-Albumin: I. Effects of pH and Ionic Strength. Int. J. Biol. Macromol. 1990, 12, (4), 263268. 59. Shang, W.; Nuffer, J. H.; Muniz-Papandrea, V. A.; Colon, W.; Siegel, R. W.; Dordick, J. S. Cytochrome c on Silica Nanoparticles: Influence of Nanoparticle Size on Protein Structure, Stability, and Activity. Small 2009, 5, (4), 470-476. 60. Robertson, W. G. Measurement of Ionized Calcium in Biological Fluids. Clin. Chim. Acta 1969, 24, (1), 149-157. 61. Brini M, Ottolini D, Calì T, Carafoli E (2013). "Chapter 4. Calcium in Health and Disease". In Sigel A, Helmut RK. Interrelations between Essential Metal Ions and Human Diseases. Metal Ions in Life Sciences. 13. Springer. pp. 81–137. 62. Saroff, H.; Lewis, M. The binding of calcium ions to serum albumin. The Journal of Physical Chemistry 1963, 67, (6), 1211-1216. 63. Xiong, Y. L. L. Influence of pH and Ionic Environment on Thermal Aggregation of Whey Proteins. J. Agric. Food Chem. 1992, 40, (3), 380-384. 64. Fordtran, J. S.; Locklear, T. W. Ionic Constituents and Osmolality of Gastric and SmallIntestinal Fluids after Eating. Am J Dig Dis 1966, 11, (7), 503-521. 19 ACS Paragon Plus Environment


513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553

65. Fallingborg, J. Intraluminal pH of the human gastrointestinal tract. Danish Medical Bulletin 1999, 46, (3), 183-196. 66. Hermansen, L.; Osnes, J. B. Blood and Muscle pH after Maximal Exercise in Man. J. Appl. Physiol. 1972, 32, (3), 304-308. 67. Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Viscoelastic acoustic response of layered polymer films at fluid-solid interfaces: Continuum mechanics approach. Phys. Scr. 1999, 59, (5), 391-396. 68. Oliva, F. Y.; Avalle, L. B.; Camara, O. R.; De Pauli, C. P. Adsorption of human serum albumin (HSA) onto colloidal TiO2 particles, Part I. J. Colloid Interface Sci. 2003, 261, (2), 299311. 69. Jones, K. L.; O'Melia, C. R. Protein and humic acid adsorption onto hydrophilic membrane surfaces: effects of pH and ionic strength. J. Membr. Sci. 2000, 165, (1), 31-46. 70. Norde, W.; Lyklema, J. Adsorption of Human-Plasma Albumin and Bovine Pancreas Ribonuclease at Negatively Charged Polystyrene Surfaces .1. Adsorption Isotherms. Effects of Charge, Ionic-Strength, and Temperature. J. Colloid Interface Sci. 1978, 66, (2), 257-265. 71. Dockal, M.; Carter, D. C.; Ruker, F. Conformational transitions of the three recombinant domains of human serum albumin depending on pH. J. Biol. Chem. 2000, 275, (5), 3042-3050. 72. Rosenoer, V. M.; Oratz, M.; Rothschild, M. A., Albumin: Structure, function and uses. Elsevier: 2014. 73. Tsai, D. H.; DelRio, F. W.; Keene, A. M.; Tyner, K. M.; MacCuspie, R. I.; Cho, T. J.; Zachariah, M. R.; Hackley, V. A. Adsorption and Conformation of Serum Albumin Protein on Gold Nanoparticles Investigated Using Dimensional Measurements and in Situ Spectroscopic Methods. Langmuir 2011, 27, (6), 2464-2477. 74. Leonard, W. J.; Foster, J. F. Changes in optical rotation in the acid transformations of plasma albumin. Evidence for the contribution of tertiary structure to rotatory behavior. J. Biol. Chem. 1961, 236, (10), 2662-2669. 75. Amirbahman, A.; Olson, T. M. The Role of Surface Conformations in the Deposition Kinetics of Humic Matter-Coated Colloids in Porous-Media. Colloid Surface A 1995, 95, (2-3), 249-259. 76. Amirbahman, A.; Olson, T. M. Transport of Humic Matter-Coated Hematite in PackedBeds. Environ. Sci. Technol. 1993, 27, (13), 2807-2813. 77. Docter, D.; Westmeier, D.; Markiewicz, M.; Stolte, S.; Knauer, S. K.; Stauber, R. H. The nanoparticle biomolecule corona: lessons learned - challenge accepted? Chem. Soc. Rev. 2015, 44, (17), 6094-6121. 78. Mao, H. Y.; Chen, W.; Laurent, S.; Thirifays, C.; Burtea, C.; Rezaee, F.; Mahmoudi, M. Hard corona composition and cellular toxicities of the graphene sheets. Colloid Surface B 2013, 109, 212-218. 79. Mirshafiee, V.; Kim, R.; Park, S.; Mahmoudi, M.; Kraft, M. L. Impact of protein precoating on the protein corona composition and nanoparticle cellular uptake. Biomaterials 2016, 75, 295-304.


Page 20 of 26

Page 21 of 26


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



Page 22 of 26

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


Page 23 of 26


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



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

Page 24 of 26

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


Page 25 of 26


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.



637

TOC Art

638


Page 26 of 26

Adsorption of Human Serum Albumin on Graphene Oxide

Recommend Documents