Formation of protein corona on nanoparticles with digestive enzymes

Subscriber access provided by EKU Libraries

Food Safety and Toxicology

Formation of protein corona on nanoparticles with digestive enzymes in simulated gastrointestinal fluids Yihui Wang, Man Li, Xingfeng Xu, Wenting Tang, Liu Xiong, and Qingjie Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05702 • Publication Date (Web): 05 Feb 2019 Downloaded from http://pubs.acs.org on February 7, 2019

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 43

Journal of Agricultural and Food Chemistry

1

Formation of protein corona on nanoparticles with digestive enzymes

2

in simulated gastrointestinal fluids

3

Yihui Wang, Man Li, Xingfeng Xu, Wenting Tang, Liu Xiong, Qingjie Sun*

4

College of Food Science and Engineering, Qingdao Agricultural University (Qingdao,

5

Shandong Province, 266109, China)

6

*Correspondence author (Tel: 86-532-88030448, Fax: 86-532-88030449, e-mail:

7

[email protected])

ACS Paragon Plus Environment


8

ABSTRACT: The protein corona (PC) that defines the biological identity of

9

nanoparticles in the blood is well known, but no comprehensive and systematic study

10

has been conducted yet on the formation of PC in the gastrointestinal environment.

11

Thus, this study aimed to explore the interaction between model polystyrene

12

nanoparticles (PS-NPs) with 50−100 nm and three digestive enzymes, namely, pepsin,

13

α-amylase, and trypsin. Results showed that the thickness of the PC formed by

14

α-amylase and trypsin was 25−100 and 50−100 nm, respectively. The zeta potential

15

values of PS-NPs after incubation significantly increased. The fluorescence quenching

16

and ultraviolet visible absorption spectra suggested the interaction between the

17

nanoparticles and the enzymes occurred. Synchronous fluorescence spectra showed

18

that the PS-NPs could induce the microenvironmental change in digestive enzymes.

19

The thermodynamic parameters suggested that the interaction was mainly driven by

20

the hydrogen bonds and van der Waals forces.

21

KEYWORDS: nanoparticle, enzyme, protein corona, interaction, gastrointestinal

22

fluids


Page 2 of 43

Page 3 of 43


23

INTRODUCTION

24

Given their small size, nanoparticles (NPs) possess superior performance, making

25

them remarkable candidates for biomedical and biotechnological applications.1-2

26

When NPs are exposed to a physiological environment such as blood, protein

27

immediately adsorbs onto their surface, leading to the formation of what is called the

28

protein corona (PC).3 The PC is commonly made up of two parts, namely, a hard

29

portion and a soft portion. The hard corona is generally composed of proteins with

30

high affinity that tightly adsorb to the bare NP surface and can prevent the adsorption

31

of other molecules, whereas the soft corona consists of loosely bound proteins with

32

low affinity and can dynamically exchange with other proteins in a solution.4-5

33

The PC usually changes the size, charge, and other structural properties of

34

nanomaterials, giving rise to a biological identity that differs from their primary

35

identity, which strongly affects their physiological response in biological systems and

36

determines the in vivo ultimate fate of particles.6 Therefore, a deep understanding of

37

the interplay between NPs and protein can undeniably benefit the study of in vivo

38

applications of NPs.7-8 At present, many studies have been performed on the

39

interactions between NPs and protein and the biological behaviors of NPs when NPs

40

are administrated in the bloodstream.9 For example, plasma protein attach onto the

41

surface of gold nanoparticles (AuNPs), forming a PC that can improve the drug

42

delivery capability of AuNPs and dictating the molecular targeting and biodistribution

43

of the NPs.10 Melby et al.11 found that protein–AuNP complexes formed in plasma

44

changed the electrokinetic, hydrodynamic, and plasmonic properties of the AuNPs. In

45

addition, the formation of a hard PC on the surface of the designed lipid NPs offered a

46

new diagnostic technology for the early detection of pancreatic cancer based on the

47

exploitation of the interaction between NPs and protein.12



48

To date, the focus on the use of nanocarriers in medicine has mostly been on

49

parenteral administration. Given the excellent performance of NPs in improving the

50

oral bioavailability of anticancer drugs and therapeutic peptides,13 exploiting the

51

behavior of NPs and their interactions with biological molecules in gastrointestinal

52

conditions is important.14 Nevertheless, our knowledge about the nano–bio interaction

53

of orally administered NPs in the gastrointestinal tract remains limited. Berardi et al9

54

reported that viral nanoparticles exposed to pig gastric and intestinal fluids were not

55

subject to protein adsorption, with no formation of a detectable PC. However, when

56

magnetite NPs were exposed to simulated digestion simultaneously with bread, PC

57

NPs were isolated from gastric and duodenal phases with different size, surface

58

charge and protein corona composition.15 Silver NPs (AgNPs) can form a PC with

59

pepsin in a simulated gastric fluid, which facilitates the aggregation of NPs and

60

induces minor variations in the pepsin tertiary structure.16 In addition, the

61

hydrodynamic diameter of zinc oxide nanoparticles remarkably increased because of

62

the formation of a PC under simulated gastric and intestinal conditions.17

63

However, previous studies are not enough to make us fully understand the

64

morphological characteristics of a PC and whether the interaction between the NPs

65

and the different enzymes occurs in simulated gastric or intestinal fluids. Even more

66

regrettable, whether the PC that forms in the gastrointestinal fluids consists of hard

67

and soft coronas like those in the blood has not been reported.

68

To address the above problems, we selected three major digestive enzymes to

69

preliminarily explore the formation of a PC and the interactions between polystyrene

70

nanoparticles (PS-NPs) and digestive enzymes, including pepsin, α-amylase, and

71

trypsin, in a simulated digestive fluid in vitro containing gastric fluids and intestinal

72

fluids. PS-NPs, acting as model NPs, were selected for this study because of their


Page 4 of 43

Page 5 of 43


73

uniformity of shape and stability in complex gastrointestinal fluids.18 The PC

74

morphology around the NPs was visualized using transmission electron microscopy

75

(TEM). We separated the soft corona from the hard corona through the centrifugal

76

washing process and monitored the evolution of the corona after each washing step

77

using TEM and dynamic light scattering (DLS). Then, we quantified the amount of

78

adsorbed protein covering the NPs by PierceTM BCA Protein Assay Kit. The

79

enzyme-NP interactions were evaluated by measuring the changes in protein

80

conformation and the protein fluorescence quenching in the presence of particles.

81

Finally, the interaction forces between the NPs and proteins were further explored by

82

thermodynamic parameters, including the free energy change (ΔG), enthalpy change

83

(ΔH), and entropy change (ΔS) of the reaction.

84

MATERIALS AND METHODS

85

Chemicals and Biochemicals. The PS-NPs (diameter = 0.05–0.1µm) were

86

purchased from Macklin Co. Ltd. (Shanghai, China). Pepsin (P7125) from porcine

87

gastric mucosa, porcine pancreatic α-amylase (A3176), and trypsin (T4799) were

88

obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Pierce™ BCA

89

protein assay kits were purchased from Thermo Fisher Scientific Inc. (Canoga Park,

90

CA, USA). All reagents used were of analytical grade.

91

Formation of the Protein Corona. The PS-NPs were incubated with simulated

92

human gastrointestinal digestion fluids to form the PC. Simulated gastric fluid (SGF)

93

and simulated intestinal fluid (SIF) were prepared according to the method of Berardi

94

et al.19 with minor modification. Briefly, 1 mL of 0.25 mg/ml PS-NP dispersion was

95

mixed with 5 ml of simulated gastric fluid (SGF, 2 mg/ml NaCl, 3.2 mg/ml pepsin,

96

and pH 1.5), and the pH of the mixture was checked and, if necessary, adjusted to 1.5



Page 6 of 43

97

with 1 M HCl. Then, the complex was further incubated at 37 °C for 30 min with

98

continuous shaking. Similarly, the same PS-NPs were incubated with the simulated

99

intestinal fluid (SIF, 6.8 mg/ml KH2PO4, 3.2 mg/ml α-amylase or 5 mg/ml trypsin,

100

and pH 6.8) under mechanical shaking for 60 min at 37 °C, maintaining a solution pH

101

of 6.8. After incubation, the above three types of suspensions were centrifuged for 20

102

min at 12,000 rpm. The pellets were then washed three times with phosphate buffer

103

solution

104

α-amylase-coated PS-NPs, and trypsin-coated PS-NPs, respectively, and then

105

freeze-dried under vacuum. These protein-coated NPs were submitted to Fourier

106

transform infrared spectroscopy (FTIR) and Pierce BCA protein assay.

to

remove

unbound

proteins,

obtaining

pepsin-coated

PS-NPs,

107

Transmission Electron Microscopy. To directly confirm the adsorption of

108

different digestive enzymes to the PS-NPs, the morphologies of the enzyme-coated

109

PS-NPs were observed using a Hitachi 7700 transmission electron microscope

110

(Tokyo, Japan). Before analysis, a drop of aqueous suspension after incubation was

111

dispensed onto carbon-coated copper grids without negative staining and dried at −60

112

ºC for TEM observations. To examine the change in the PC formed on the surface of

113

PS-NPs, the samples gained by centrifugation and each washing step were also

114

observed with TEM.

115

DLS Measurements. The size distribution and average diameter of the samples

116

were examined by DLS,18 which was performed with a commercially available

117

instrument, namely, the Malvern Zetasizer Nano instrument (Malvern Instruments

118

Ltd., Malvern, U.K.), equipped with a helium–neon laser (0.4 mW, 633 nm). The bare

119

PS-NPs and protein-coated PS-NPs were diluted to 1.0 mg/ml, measured in a cuvette

120

and equilibrated at 25 ± 1 °C prior to analysis.


Page 7 of 43


121

Zeta Potential Measurements. A Malvern zeta sizer nano series instrument was

122

used to measure the zeta (ζ) potential of the bare PS-NPs, digestive enzymes, and the

123

NP–protein complex. The complex was prepared as described above. The zeta

124

potential of the bare NPs was determined in the SGF and SIF without digestive

125

enzymes. To measure the zeta potential of the different enzymes, pepsin was

126

dissolved in SGF, and α-amylase and trypsin were dissolved in SIF.

127

Pierce BCA Protein Quantification Assay. The protein content of the three

128

different types of hard PC was assessed using the BCA protein assay based on the

129

manufacturer's instructions. The protein concentration in each experiment was

130

detected at 562 nm.

131

FTIR Spectroscopy Analysis. The FTIR spectra of the freeze-dried PS-NPs,

132

digestive enzymes, and protein-coated NPs were recorded by an FTIR

133

spectrophotometer (NEXUS-870; Thermo Nicolet Corporation, Madison, WI, USA).

134

The spectra were collected from an accumulation of 64 scans with a 4 cm−1 resolution

135

between 4,000 cm-1 and 400 cm-1 in the transmittance mode.

136

Ultraviolet-visible (UV-vis) Absorption Spectra. The concentration dependence

137

of the PS-NP–protein interactions was analyzed using a UV-vis spectrophotometer

138

(TU-1810, Beijing, China) in the wavelength range of 200–350 nm. A series of

139

sample solutions was prepared by maintaining the enzyme concentration (3.2 mg/ml

140

of pepsin, 3.2 mg/ml of α-amylase, and 5 mg/ml of trypsin) and increasing the

141

concentrations of PS-NPs to 0–0.1 mg/ml. The final absorption spectra were baseline

142

corrected by deionized water.

143

Fluorescence Spectroscopy Measurements. Fluorescence spectra of the

144

different types of digestive enzymes with PS-NPs were obtained by an F-7000



145

(Hitachi, Japan) spectrofluorimeter. Various concentrations of PS-NPs (0, 0.01, 0.03,

146

0.05, 0.07, and 0.1 mg /mL) were incubated for 20 min at room temperature with

147

pepsin (3.2 mg/ml), α-amylase (3.2 mg/ml), and trypsin (5 mg/ml), respectively.

148

Then, 3 mL of the sample was subjected to analysis at the excitation wavelength of

149

280 nm and with emission spectra in the 280–420 nm range.

150

Statistical Analysis. All measurements were conducted at least in triplicate. The

151

statistical analysis was conducted using the Statistical Package for the Social Sciences

152

version 17.0 (SPSS Inc., Chicago, IL, USA). Duncan's multiple range test was

153

performed to analyze the difference in means from ANOVA at a significance level of

154

5% (p < 0.05).

155

RESULTS AND DISCUSSION

156

Formation of the Protein Corona. To investigate the evolution of the PC after

157

the incubation of PS-NPs with each digestive enzyme, we characterized the

158

morphologies and size development of the NP–protein complex by TEM and DLS.

159

Directly after the incubation of NPs in the SGF-containing pepsin, SIF-containing

160

α-amylase, and SIF-containing trypsin (Figure 1), the PS-NPs were surrounded by a

161

cloud of protein, and the diameter of the protein-coated NPs was larger than that of

162

the bare NPs with same size, observed by TEM. The PC on the surface of the PS-NPs

163

for pepsin (Figure 1A ) was formed by the aggregation of super small pepsin NPs.

164

The TEM image of pepsin showed a uniform spherical shape at a size of 7 ± 2.0 nm

165

(Figure S1 ), and the hydrodynamic diameter by DLS was measured at 15±2.5 nm

166

(Figure S2). The size distribution after incubation through the DLS was bimodal,

167

where the smaller peak corresponds to the size of the pepsin particles and the larger

168

peak represents the size of the NP-pepsin complex and the aggregated PS-NPs. The


Page 8 of 43

Page 9 of 43


169

formation of PC can increase the diameter of NPs, but through TEM, the aggregation

170

of NPs was also observed. Therefore, the increase of NPs size obtained by DLS could

171

also be due to some partial aggregation of NPs. An incremental separation process

172

was conducted to differentiate between the soft corona and the hard corona. The

173

PS-NPs were still coated with a vast amount of protein as observed after the first

174

centrifugation. After the first washing step, the thickness of the protein corona

175

decreased as most of the soft corona was removed. However, the hydrodynamic size

176

of NPs did not reduce, which may be caused by the aggregation of NPs. The NP–

177

pepsin complex with the hard PC was obtained through three times of washing.

178

When the PS-NPs were incubated with α-amylase (Figure 1B) and trypsin

179

(Figure 1C), a thick homogeneous PC formed on the NP surfaces, and it was

180

remarkably different from that of the pepsin-coated NPs. The visible thickness of

181

α-amylase on the PS-NPs was about 25–100 nm ,and the thickness of trypsin on the

182

PS-NPs was about 50–100 nm, observed by TEM. Meanwhile, there was visible

183

aggregation in TEM images after incubation，especially for α-amylase. Therefore，the

184

hydrodynamic diameter of NPs determined by DLS increased, which may be caused

185

by the combined action of the formed PC and the aggregation of nanoparticles. After

186

centrifugation and three times of washing, the thickness of the corona formed by both

187

α-amylase and trypsin decreased and became non-uniform as the soft PC was washed

188

away. However, the hydrodynamic size of the α-amylase-coated PS-NPs and the

189

trypsin-coated PS-NPs gained from the DLS did not decrease gradually with the

190

washing process. This reaction could be attributed to the aggregation of NPs caused

191

by centrifugation. The PC that existed on the surface of the PS-NPs did not have

192

significant morphological differences whether the complex was washed one time or

193

three times.



194

The formation of a PC on the surfaces of the PS-NPs was further confirmed by the

195

surface charge change. The zeta potential measurements of the bare PS-NPs, pure

196

enzymes, and NP complex formed with each enzyme were performed. Figure. 2

197

shows that the surface zeta potential of the bare PS-NPs was −10 ± 1.2 in the SGF

198

without pepsin and −27 ±1.3 mV in the SIF without α-amylase or trypsin,

199

respectively. After the NPs were incubated with pepsin, α-amylase, and trypsin, the

200

values of the zeta potential increased significantly, showing a negative surface charge

201

of −3 ± 0.9, −18 ± 1.4, and −17 ± 1.3 mV. These results suggest that the NPs were

202

covered with a PC on their surfaces. Zhang et al.20 reported that the size of the

203

ligand-modified NPs with an in vitro PC significantly increased and that the zeta

204

potential of the NPs changed from positive to slightly negative, indicating the

205

adsorption of the proteins with a negative potential around the NPs.

206

Quantitative Analysis of the Protein Corona Formation on the Surface of the

207

PS-NPs. To determine the amount of strongly adsorbed proteins, the so-called hard

208

protein coronas,21-22 on the surface of the NPs, a protein quantity assay was conducted

209

after three centrifugation steps. The amount of hard corona proteins on the NPs was

210

determined by the Pierce BCA protein assay. The amount of adsorbed proteins per

211

microgram of NPs is shown in Figure. 3. For pepsin and α-amylase, the values of 104

212

± 0.12 μg/mg and 107 ± 0.18 μg/mg, respectively, were obtained. For trypsin, the

213

value decreased to 16.14 ± 0.06 μg/mg. The results indicate that the PS-NPs had a

214

weak adsorption capacity for trypsin but a strong adsorption capacity for pepsin and

215

amylase. Zhang et al20 reported that the NPs modified by the transferrin

216

receptor-targeting ligand had a strong adsorption capacity for human plasma protein

217

of up to 100 μg/mg.

218

Investigation of protein–particle interactions


Page 10 of 43

Page 11 of 43


219

FTIR Spectroscopy. The FTIR spectra of the three native digestive enzymes and

220

the enzyme–NP complexes were investigated to identify the structural changes of

221

enzymes caused by the interaction between PS-NPs and digestive enzymes (Figure 4).

222

The amide band of protein mainly comprises amide I, which is based on the stretching

223

vibrations of C=O and C–N, and amide II, which is attributed to the combination of

224

the N–H in-plane bending and C–N stretching vibrations of the peptide groups23. The

225

FTIR spectrum of native pepsin displayed two characteristic bands at 1652 cm-1 and

226

1438 cm-1, which corresponded to C=O stretching and C-N stretching , respectively.24

227

However, in the spectra of the NP–pepsin complex, we observed that the frequency

228

belonging to the C=O stretching and C-N stretching shifted to 1631 cm-1 and 1397

229

cm-1, respectively. The results suggested that the addition of PS-NPs caused the

230

transformation in the second structure of pepsin. Similarly, Wang et al.25 found that

231

when AuNPs were added to transferrin, the amide bonds of the protein altered,

232

leading to changes in the secondary structure. Furthermore, the C-N stretching

233

vibration at 2932 cm-1 that originated from the methylene groups shifted to the short

234

wavelength of 2926 cm-1 because of the interaction between NPs and pepsin.

235

For α-amylase and trypsin, the variation of the FTIR spectra before and after

236

combining with the NPs was similar to the changes in pepsin. The characteristic peak

237

at 1632 cm-1 based on amide I, whether α-amylase or trypsin, had a shift to 1636 cm-1

238

and 1644 cm-1, respectively. The amide II of α-amylase and trypsin was shifted from

239

1424 cm-1 to 1397 cm-1 and 1438 cm-1 to 1371 cm-1, respectively. The band at 2933

240

cm-1 of both α-amylase and trypsin was shifted to 2925 cm-1 upon interacting with the

241

PS-NPs, indicating the structural change of the enzyme. Jiang et al.26 reported that the

242

change in the secondary structural conformation of α-amylase was observed through

243

the alteration of the feature bands, including 1625cm-1 (amide I), 1520 cm-1 (amide



244

II), and 2927 cm-1 (C–H stretching ), and it was attributed to the interaction between

245

α-amylase and starch NPs. Furthermore, compared with pure PS-NPs, a series of

246

characteristic peaks of protein corresponding to the native enzyme appeared at 1,000

247

cm-1–1,100 cm-1 for the three types of enzyme–NP mixtures. The result also proved

248

that protein adsorbed on the surface of the PS-NPs and that an interaction between the

249

enzyme and the PS-NPs occurred, consistent with the finding of Yang et al.27

250

UV–vis Spectra. The UV–vis absorption spectra are considered a simple and

251

effective method to examine the interaction of enzyme with NPs and the structural

252

transitions in enzyme.28 The UV-vis absorption spectra of pepsin, α-amylase, and

253

trypsin in the absence and presence of PS-NPs are shown in Figure 5. Clearly, with

254

the addition of NPs, the absorption peak of trypsin at 279 nm and at 220 nm

255

significantly increased (Figure 5C). The strong absorption peak at 220 nm represents

256

the absorption peak of a typical peptide bond backbone structure of protein, and the

257

weak absorption peak of trypsin at around 279 nm is ascribed to the aromatic amino

258

acids (Trp, Tyr, and Phe).29 With the increasing concentration of PS-NPs, the peak

259

intensity of trypsin at 220 nm increased with a red shift, and the intensity of the peak

260

at 279 nm increased with a slight blue shift. This result suggests that the interaction

261

between PS-NPs and trypsin led to the loosening and unfolding of the protein

262

backbone and reduced the hydrophobicity of the microenvironment of trypsin.

263

Similarly, the changes in the UV-vis absorption spectra of pepsin (Figure 5A) and

264

α-amylase (Figure 5B) have the same rule as those in trypsin, which also indicates

265

that pepsin and α-amylase interacted with PS-NPs. Li et al.30 reported that an

266

interaction occurred between the AgNPs and pepsin using a UV-vis spectrometer. Ji

267

et al.31 found that the UV–vis absorption spectra of α-amylase enhanced successively

268

when the concentration of cellulose nanocrystals gradually increased, thus suggesting


Page 12 of 43

Page 13 of 43

269


the interaction between cellulose nanocrystals and α-amylase.

270

Fluorescence Spectra of Digestive Enzyme with PS-NPs. The interaction between

271

PS-NPs and pepsin, α-amylase, and trypsin was further determined by fluorescence

272

measurement because of the intrinsic fluorescence in proteins.32 The fluorescence

273

intensity of pepsin, α-amylase, and trypsin had a remarkable progressive decrease as

274

the particle concentration increased, indicating the quenching of protein fluorescence

275

induced by the PS-NPs. The results showed the formation of the enzyme–NP complex

276

and the occurrence of the interaction between the enzyme and the NPs. The maximum

277

emission wavelength of pepsin (Figure 6A) and trypsin (Figure 6C) had no obvious

278

shift. However, a slight shift in the maximum emission wavelength occurred from

279

α-amylase, indicating that the microenvironment around the chromophore of

280

α-amylase altered because of the action of the PS-NPs. Sun et al.33 reported that the

281

fluorescence intensity of the human serum albumin (HSA) decreased with the

282

addition of TiO2 NPs and that the maximum emission wavelength shifted, indicating

283

the occurrence of the interaction between HSA and TiO2 NPs.

284

Synchronous Fluorescence Spectra. The variation in the synchronous fluorescence

285

spectra can reflect the change in the microenvironment surrounding tyrosine or

286

tryptophan residues in the 3D structure of protein. When the synchronous

287

fluorescence spectra were measured at Δλ = 15 nm or 60 nm, the characteristic

288

information of tyrosine residues or tryptophan residues was gained separately from

289

pepsin, a-amylase, and trypsin. Figure 7 shows the synchronous fluorescence spectra

290

of pepsin, α-amylase, and trypsin with the increasing concentration of the PS-NPs.

291

Clearly, the binding of PS-NPs to pepsin, α-amylase, and trypsin caused the

292

fluorescence quenching of Tyr and Trp residues, indicating that Tyr and Trp residues

293

affected the binding interactions of the PS-NPs with the digestive enzymes. For



Page 14 of 43

294

pepsin, as shown in Figure 7A, the addition of PS-NPs resulted in a dramatic decline

295

in the fluorescence intensity. Moreover, the maximum emission wavelength was not

296

significantly changed at Δλ = 15 but slightly shifted at Δλ = 60 nm from 279 nm to

297

277 nm, suggesting the increase in hydrophobicity around the Tyr and Trp residues.

298

The fluorescence spectra of the residues in α-amylase (Figure 7B) almost had no shift,

299

indicating that the polarity around the Tyr and Trp residues was still reserved.

300

Similarly, we found that the hydrophobicity around the Tyr and Trp residues in

301

trypsin decreased because of the occurrence of a red shift (Figure 7C). Chen et al.34

302

reported that the fluorescence intensities of both Trp and Tyr residues in HSA

303

decreased with the addition of isorenieratene and that the maximum emission

304

wavelength had a red shift, indicating the increase in polarity of the Trp and Tyr

305

residues.

306

Fluorescence Quenching Mechanism. To further elucidate the fluorescence

307

quenching mechanism caused by the PS-NPs, the Stern–Volmer equation was used to

308

process the quenching data35:

309

F0/F = 1 + KSV [Q] = 1 + kqτ0[Q]

310

where F0 and F are the fluorescence intensities of pepsin, α-amylase, and trypsin in the

311

absence and presence of the PS-NPs, respectively. KSV is the quenching constant, kq

312

is the bimolecular quenching rate constant, τ0 is the average lifetime of the molecule,

313

which is about 10-8 S, and [Q] is the concentration of the PS-NP quencher.

(1)

314

The Stern–Volmer quenching plots of pepsin, α-amylase, and trypsin with the

315

PS-NPs at different temperatures (298, 304, and 310 K) are shown in Figure 8. We

316

observed a good linear behavior between F0/F and [Q], suggesting a single dynamic

317

quenching or static quenching mechanism. The Ksv values, presented in Table 1, for


Page 15 of 43


318

the pepsin–NP complex and amylase–NP complex decreased with increasing

319

temperature, indicating that the quenching mechanism of pepsin and α-amylase by the

320

PS-NPs was static. On the contrary, the quenching constant Ksv of the trypsin–NP

321

complex increased with increasing temperature, indicating that dynamic quenching

322

occurred in the trypsin and PS-NPs system. Guo et al.36 estimated that the

323

fluorescence quenching of HSA by LaPO4:Eu nanorods followed a dynamic

324

mechanism because Ksv increased with increasing temperature. In addition, the

325

binding site numbers (n) were approximately equal to 1 in the different temperatures,

326

suggesting that that there was roughly one binding site in pepsin, α-amylase, and

327

trypsin separately for the PS-NPs.

328

Thermodynamic Parameters. The type of binding force can be determined by the

329

thermodynamic parameters. The thermodynamic parameters (∆G, ∆H, and ∆S) were

330

estimated by the van’t Hoff equation, as shown in Figure 9:

331

ln ka  

332

G  H - TS

333

where ΔH, ΔG, and ΔS are the enthalpy change, free enthalpy change, and entropy

334

change, respectively, and Ka is the binding constant at 298, 304, and 310 K. R is the

335

gas constant, and T is the experimental temperature.

H S  RT RT

(3)

(4)

336

The negative values of the free energy change (∆G) and the enthalpy change

337

(∆H) (Table 1) in the different temperatures indicated that the interaction between the

338

NPs and the digestive enzyme was spontaneous and thermopositive. The ∆H can

339

represent the increase in intermolecular bond energies, and ∆S can denote a

340

disordered change in the system during the binding process. There are four main



341

banding forces between the NPs and proteins, namely, hydrogen bonding,

342

electrostatic interaction, van der Waals interaction, and hydrophobic force, which can

343

be characterized by the sign and magnitude of the thermodynamic parameter.34 As

344

shown in Table1, the negative ΔH and ΔS indicated that the binding between the

345

PS-NPs and the digestive enzyme was mainly driven by the hydrogen bond and the

346

van der Waals forces.

347

In summary, model PS-NPs were covered with digestive enzymes after

348

incubation in a gastrointestinal environment to form a layer of PC. The PC formed by

349

pepsin consisted of small spherical particles. Its morphology changed obviously, and

350

only a thin layer of protein remained on the surface of the PS-NPs after three washing

351

step. The PS-NPs were covered with a thick and uniform PC after incubation in

352

α-amylase or trypsin. The morphology of the corona did not change, but the thickness

353

decreased after the washing step.

354

Accompanied by the formation of the corona, the size and the surface charge of

355

the NPs were altered. According to the results of the quantitative analysis, the affinity

356

of the PS-NPs to pepsin and α-amylase was higher than that to pepsin. In addition, the

357

PS-NPs could induce the fluorescence quenching of pepsin, α-amylase, and trypsin

358

and change the secondary structure. Furthermore, the complexation process could be

359

driven by the hydrogen bond or the van der Waals forces according to the

360

thermodynamic parameter (∆H < 0, ∆S < 0), which implies that the electrostatic

361

interaction is not the only driving force of the interaction between the particles and

362

protein. The enzyme-coated NPs are suggested to present a completely different

363

behavior compared with the bare NPs in gastrointestinal conditions, as they exhibit an

364

important effect on the availability of NPs as nanocarriers. Therefore, our results

365

emphasize that exploring the interaction of the NPs and protein is crucial to facilitate


Page 16 of 43

Page 17 of 43


366

the prediction of the protein adsorption on the NPs and their behavior in the

367

gastrointestinal system. Further study is required to evaluate the interaction of

368

food-grade NPs with proteins in the gastrointestinal tract.



369

NOTES

370

The authors declare no competing financial interest.

371

ACKNOWLEDGMENTS

372

The study was supported by the National Natural Science Foundation, China (Grant

373

No. 31671814), Major Agricultural Application Technology Innovation Project of

374

Shandong Province (Project No. SF1405303301), and Special Funds for Taishan

375

Scholars Project of Shandong Province (No. ts201712058).


Page 18 of 43

Page 19 of 43


376

REFERENCE

377

1.

378

nanomaterials with proteins in a physiological environment. Chemical Society

379

Reviews 2012, 43, 2780-2799.

380

2.

381

Visualization of the protein corona: towards a biomolecular understanding of

382

nanoparticle-cell-interactions. Nanoscale 2017, 9, 8858-8870.

383

3.

384

of Protein Conformational Change and Binding Affinity in Protein-Nanoparticle

385

Interaction. Analytical Chemistry 2017, 89, 12160-12167.

386

4.

387

Franzese, G., Understanding the Kinetics of Protein–Nanoparticle Corona Formation.

388

ACS nano 2016, 10, 10842-10850.

389

5.

390

I.; Weitz, D. A.; Filippov, S. K., Interaction of spin-labeled HPMA-based

391

nanoparticles

392

protein-corona-free polymer nanomedicine. Nanoscale 2018, 10, 6194-6204.

393

6.

394

Kintzel, U.; Kaltbeitzel, A.; Renz, P.; Domogalla, M.; Steinbrink, K.; Lieberwirth, I.;

395

Crespy, D.; Landfester, K.; Mailänder, V., Pre-adsorption of antibodies enables

396

targeting of nanocarriers despite a biomolecular corona. Nature nanotechnology 2018,

397

13, 862–869.

Walkey, C. D.; Chan, W. C., Understanding and controlling the interaction of

Kokkinopoulou, M.; Simon, J.; Landfester, K.; Mailänder, V.; Lieberwirth, I.,

Duan, Y.; Liu, Y.; Shen, W.; Zhong, W., Fluorescamine Labeling for Assessment

Vilanova, O.; Mittag, J. J.; Kelly, P. M.; Milani, S.; Dawson, K. A.; Rädler, J. O.;

Klepac, D.; Kostková, H.; Petrova, S.; Chytil, P.; Etrych, T.; Kereïche, S.; Raška,

with

human

blood

plasma

proteins

-

the

introduction

of

Tonigold, M.; Simon, J.; Estupiñán, D.; Kokkinopoulou, M.; Reinholz, J.;



398

7.

Charchar, P.; Christofferson, A. J.; Todorova, N.; Yarovsky, I., Understanding

399

and Designing the Gold-Bio Interface: Insights from Simulations. Small 2016, 12,

400

2395-2418.

401

8.

402

of Biocompatible Inorganic Nanoparticles Towards Biomedical Applications.

403

Biomaterials Science 2017, 6, 726-745

404

9.

405

virus-based nanocarriers in gastrointestinal fluids. Nanoscale 2017, 10, 1667-1679.

406

10. Charbgoo, F.; Nejabat, M.; Abnous, K.; Soltani, F.; Taghdisi, S. M.; Alibolandi,

407

M.; Thomas, W. S.; Steele, T.; Ramezani, M., Gold nanoparticle should understand

408

protein corona for being a clinical nanomaterial. Journal of Controlled Release

409

Official Journal of the Controlled Release Society 2018, 272, 39-53.

410

11. Melby, E. S.; Lohse, S. E.; Park, J. E.; Vartanian, A. M.; Putans, R. A.; Abbott,

411

H. B.; Hamers, R. J.; Murphy, C. J.; Pedersen, J. A., Cascading Effects of

412

Nanoparticle Coatings: Surface Functionalization Dictates the Assemblage of

413

Complexed Proteins and Subsequent Interaction with Model Cell Membranes. ACS

414

nano 2017, 11, 5489-5499.

415

12. Caputo, D.; Papi, M.; Coppola, R.; Palchetti, S.; Digiacomo, L.; Caracciolo, G.;

416

Pozzi, D., A protein corona-enabled blood test for early cancer detection. Nanoscale

417

2017, 9, 349-354.

418

13. Garcã-A-Dã-Az, M.; Birch, D.; Wan, F.; Nielsen, H. M., The role of mucus as an

419

invisible cloak to transepithelial drug delivery by nanoparticles. Adv Drug Deliv Rev

420

2017, 124, 107-124.

Jiao, M.; Zhang, P.; Meng, J.; Li, Y.; Liu, C.; Luo, X.; Gao, M., Recent Advances

Berardi, A.; Evans, D. J.; Baldelli, F. B.; Lomonossoff, G. P., Stability of plant


Page 20 of 43

Page 21 of 43


421

14. Bouwmeester, H.; Van, d. Z. M.; Jepson, M. A., Effects of food-borne

422

nanomaterials on gastrointestinal tissues and microbiota. Wiley Interdiscip Rev

423

Nanomed Nanobiotechnol 2017, 10.

424

15. Di, S. D.; Rigby, N.; Bajka, B.; Mackie, A.; Bombelli, F. B., Effect of protein

425

corona magnetite nanoparticles derived from bread in vitro digestion on Caco-2 cells

426

morphology and uptake. International Journal of Biochemistry & Cell Biology 2016,

427

75, 212-222.

428

16. Ault, A. P.; Stark, D. I.; Axson, J. L.; Keeney, J. N.; Maynard, A. D.; Bergin, I.

429

L.; Philbert, M. A., Protein Corona-Induced Modification of Silver Nanoparticle

430

Aggregation in Simulated Gastric Fluid. Environ Sci Nano 2017, 3, 1510-1520.

431

17. Yu, J.; Kim, H. J.; Go, M. R.; Bae, S. H.; Choi, S. J., ZnO Interactions with

432

Biomatrices: Effect of Particle Size on ZnO-Protein Corona. Nanomaterials 2017, 7,

433

377.

434

18. Walczak, A. P.; Kramer, E.; Hendriksen, P. J.; Helsdingen, R.; Van, d. Z. M.;

435

Rietjens, I. M.; Bouwmeester, H., In vitro gastrointestinal digestion increases the

436

translocation of polystyrene nanoparticles in an in vitro intestinal co-culture model.

437

Nanotoxicology 2015, 9, 886-894.

438

19. Berardi, A.; Lomonossoff, G. P.; Evans, D. J.; Barker, S. A., Plant-expressed

439

Hepatitis B core antigen virus-like particles: Characterization and investigation of

440

their stability in simulated and pig gastro-intestinal fluids. International Journal of

441

Pharmaceutics 2017, 522, 147-156.

442

20. Zhang, H.; Wu, T.; Yu, W.; Ruan, S.; He, Q.; Gao, H., Ligand Size and

443

Conformation Affect the Behavior of Nanoparticles Coated with in Vitro and in Vivo

444

Protein Corona. Acs Applied Materials & Interfaces 2018, 10, 9094-9103.



445

21 Winzen, S.; Schoettler, S.; Baier, G.; Rosenauer, C.; Mailaender, V.; Landfester,

446

K.; Mohr, K., Complementary analysis of the hard and soft protein corona: sample

447

preparation critically effects corona composition. Nanoscale 2015, 7, 2992-3001.

448

22. Müller, J.; Bauer, K. N.; Prozeller, D.; Simon, J.; Mailänder, V.; Wurm, F. R.;

449

Winzen, S.; Landfester, K., Coating nanoparticles with tunable surfactants facilitates

450

control over the protein corona. Biomaterials 2017, 115, 1-8.

451

23. Zhou, Z.; Xiang, H.; Zhou, M.; Meng, F., Chemically induced alternations in the

452

characteristics of fouling-causing bio-macromolecules - Implications for the chemical

453

cleaning of fouled membranes. Water Research 2016, 108, 115-123.

454

24. Sekar, G.; Sugumar, S.; Mukherjee, A.; Chandrasekaran, N., Multiple

455

spectroscopic studies of the structural conformational changes of human serum

456

albumin—Essential oil based nanoemulsions conjugates. Journal of Luminescence

457

2015, 161, 187-197.

458

25. Wang, X.; Wang, M.; Lei, R.; Zhu, S. F.; Zhao, Y.; Chen, C., Chiral Surface of

459

Nanoparticles Determines the Orientation of Adsorbed Transferrin and Its Interaction

460

with Receptors. ACS nano 2017, 11, 4606-4616.

461

26. Jiang, S.; Li, M.; Chang, R.; Xiong, L.; Sun, Q., In vitro inhibition of pancreatic

462

α-amylase by spherical and polygonal starch nanoparticles. Food & Function 2017, 9,

463

355-363.

464

27. Yang, J.; Chang, R.; Ge, S.; Zhao, M.; Liang, C.; Xiong, L.; Sun, Q., The

465

inhibition effect of starch nanoparticles on tyrosinase activity and its mechanism.

466

Food & Function 2016, 7, 4804-4815.


Page 22 of 43

Page 23 of 43


467

28. Momeni, L.; Shareghi, B.; Saboury, A. A.; Farhadian, S.; Reisi, F., A

468

spectroscopic and thermal stability study on the interaction between putrescine and

469

bovine trypsin. International Journal of Biological Macromolecules 2017, 94,

470

145-153.

471

29. Pan, X.; Qin, P.; Liu, R.; Wang, J., Characterizing the Interaction between

472

Tartrazine and Two Serum Albumins by a Hybrid Spectroscopic Approach. J Agric

473

Food Chem 2011, 59, 6650-6656.

474

30. Li, X.; Wang, K.; Peng, Y., Exploring the interaction of silver nanoparticles with

475

pepsin and its adsorption isotherms and kinetics. Chemico-Biological Interactions

476

2018, 286, 52-59.

477

31. Ji, N.; Liu, C.; Li, M.; Sun, Q.; Xiong, L., Interaction of cellulose nanocrystals

478

and amylase: Its influence on enzyme activity and resistant starch content. Food

479

Chemistry 2018, 245, 481-487.

480

32. Cao, S.; Liu, B.; Li, Z.; Chong, B., A fluorescence spectroscopic study of the

481

interaction between Glipizide and bovine serum albumin and its analytical

482

application. Journal of Luminescence 2014, 145, 94-99.

483

33. Sun, W.; Du, Y.; Chen, J.; Kou, J.; Yu, B., Interaction between titanium dioxide

484

nanoparticles and human serum albumin revealed by fluorescence spectroscopy in the

485

absence of photoactivation. Journal of Luminescence 2009, 129, 778-783.

486

34. Chen, Y.; Zhou, Y.; Mo, C.; Xie, B.; Yang, J.; Chen, J.; Sun, Z., Isorenieratene

487

interaction with human serum albumin: Multi-spectroscopic analyses and docking

488

simulation. Food Chemistry 2018, 258, 393-399.



489

35. Das, S.; Das, A.; Maji, A.; Beg, M.; Singha, A.; Hossain, M., A compact study on

490

impact of multiplicative Streblus asper inspired biogenic silver nanoparticles as

491

effective photocatalyst, good antibacterial agent and interplay upon interaction with

492

human serum albumin. Journal of Molecular Liquids 2018, 259, 18-29.

493

36. Guo, X.; Yao, J.; Liu, X.; Wang, H.; Zhang, L.; Xu, L.; Hao, A., LaPO4:Eu

494

fluorescent nanorods, synthesis, characterization and spectroscopic studies on

495

interaction with human serum albumin. Spectrochimica Acta Part A Molecular &

496

Biomolecular Spectroscopy 2018, 198, 248-256.


Page 24 of 43

Page 25 of 43


497

FIGURE CAPTIONS

498

Figure 1. TEM images of the protein corona after PS-NPs incubated with (A) pepsin,

499

(B) α-amylase and (C) trypsin. Centrifugation 1: after centrifugation and removal of

500

the unbound protein, but before washing step. Size distribution of PS-NPs in pepsin,

501

α-amylase and trypsin respectively before and after washing steps was characterized

502

by DLS. Bare PS-NPs: as a control.

503

Figure 2. Zeta potential of PS-NPs without and with protein corona. Black: bare

504

PS-NPs. Red: digestive enzymes. Blue: NPs covered with protein corona. Data are

505

shown as the mean ± standard error of three independent experiments.

506

Figure 3. The amount of protein recovered from the surface of bare NPs after three

507

washing steps determined by BCA protein assay. Mean values of triplicates with

508

standard deviation are show.

509

Figure 4. The FTIR spectra of the PS-NPs, enzymes and enzyme-NP complexes. (A):

510

pepsin. (B): α-amylase. (C): trypsin.

511

Figure 5. The UV–Vis absorption spectra of pepsin (A), α-amylase (B) and trypsin

512

(C) incubated with various concentrations of PS-NPs. NPs concentrations from a to f:

513

0, 0.03, 0.05, 0.07, and 0.1 mg/ml, respectively. a: bare PS-NPs, 0.1 mg/ml.

514

Figure 6. The fluorescence emission spectra obtained for pepsin (A), α-amylase (B)

515

and trypsin (C) incubated with various concentrations of PS-NPs. NPs concentrations

516

from a to f: 0, 0.01, 0.03, 0.05, 0.07, and 0.1 mg/ml.

517

Figure 7. Synchronous fluorescence spectra of pepsin (A), α-amylase (B) and trypsin

518

(C) in the presence of different concentrations of PS-NPs. NPs, a - f: 0, 0.01, 0.03,

519

0.05, 0.07, and 0.1 mg/ml.



520

Figure 8. The Stern–Volmer curves of pepsin (A), α-amylase (B) and trypsin (C)

521

quenched by NPs at 298, 304, and 310 K. The error bar corresponds to the standard

522

deviation for n = 3.

523

Figure 8. The Stern–Volmer curves of pepsin (A), α-amylase (B) and trypsin (C)

524

quenched by NPs at 298, 304, and 310 K. The error bar corresponds to the standard

525

deviation for n = 3.

526

Figure 9. Van’t Hoff plots of PS-NPs interaction with pepsin (A), α-amylase (B) and

527

trypsin (C).


Page 26 of 43

Page 27 of 43


Table 1 Quenching parameters, binding constants and thermodynamic parameters for three enzyme-NP complexes at different temperatures. system Pepsin-NPs

α-amylase-NPs

Trypsin-NPs

Ksv×103

Kq×108

(L mol-1)

(L mol-1S-1)

298

0.996

0.996

304

0.900

310

T/K

n

ΔG

ΔH

ΔS

(KJ mol-1)

(KJ mol-1)

(J mol-1 K-1)

0.890

-14.74

-26.53

-39.57

0.900

0.861

-14.50

0.863

0.863

0.891

-15.04

298

1.332

1.332

0.857

-15.12

-29.45

-48.09

304

1.302

1.302

0.839

-14.83

310

1.187

1.187

0.990

-17.98

298

1.076

1.076

1.081

-18.83

-164.5

-488.90

304

1.140

1.140

0.903

-15.89

310

1.200

1.200

0.908

-16.45



A

Wash 1

Wash 2

Wash 3

Bare PS-NPs

PS-NPs Incubation with pepsin Centrifugation 1 Wash 1 Wash 2 Wash 3

25 20 Intensity

Centrifugation 1

Incubation with pepsin

30

15 10 5 0 10

Page 28 of 43

100

1000

Diamater(nm)


Page 29 of 43


B Centrifugation 1

Incubation with α-amylase

Wash 1

Wash 2

Wash 3

25

PS-NPs Incuation with trypsin Centrifugation 1 Wash 1 Wash 2 Wash 3

20

Intensity

15

10

5

0 10

100 Diameter (nm)


1000


Page 30 of 43

C Incubation with trypsin

Centrifugation 1

Wash 1

Wash 2

Wash 3

25

PS-NPs Incuation with trypsin Centrifugation 1 Wash 1 Wash 2 Wash 3

20

Intensity

15

10

5

0 10

100

1000

Diameter (nm)

Figure 1. TEM images of the protein corona after PS-NPs incubated with (A) pepsin, (B) α-amylase and (C) trypsin. Centrifugation 1: after centrifugation and removal of the unbound protein, but before washing step. Size distribution of PS-NPs in pepsin, α-amylase and trypsin respectively before and after washing steps was characterized by DLS. Bare PS-NPs: as a control.


Page 31 of 43


Pepsin

amylase

Trypsin

0

Zeta potential (mV)

-5 -10 -15 -20

PS-NPs enzyme compound

-25 -30

Figure 2. Zeta potential of PS-NPs without and with protein corona. Black: bare PS-NPs. Red: digestive enzymes. Blue: NPs covered with protein corona. Data are shown as the mean ± standard error of three independent experiments.



μg protein / mg PS-NPs

120 100 80 60 40 20 0 Pepsin

α-amylase

Trypsin

Figure 3. The amount of protein recovered from the surface of bare NPs after three washing steps determined by BCA protein assay. Mean values of triplicates with standard deviation are shown.


Page 32 of 43

Page 33 of 43


PS-NPs Pepsin Pepsin+PS-NPs

Transmittance (%)

A

1397 1631 2926 1652

1438

amide I amide II 2932

4000

3500

3000

2500

2000

1500

1000

500

-1 Wavenumber (cm )

PS-NPs α- amylase α- amylase+PS-NPs

Transmittance (%)

B

1636 2925

1397

1632

amide I 1424

amide II

2933

4000

3500

3000

2500

2000

1500

1000

-1 Wavenumber (cm )


500


PS-NPs Trypsin Trypsin+PS-NPs

C

Transmittance (%)

1644

1371

2925

1632

amide I

1438

amide II 2933

4000

3500

3000

2500

2000

1500

1000

500

-1 Wavenumber (cm )

Figure 4. The FTIR spectra of the PS-NPs, enzymes and enzyme-NP complexes. (A): Pepsin. (B): α-amylase. (C): trypsin.


Page 34 of 43

Page 35 of 43


1.6 1.4 1.2 Absorbance

1.8

a b c d e f g

A

1.2

0.8 0.6

1.0 0.8 0.6

0.4

0.4

0.2

0.2 250

300

350

0.0 200

Wavelength (nm)

1.8

250

300

Wavelength (nm)

a b c d e f g

C

1.6 1.4 1.2 Absorbance

a b c d e f g

1.4

1.0

0.0 200

B

1.6

Absorbance

1.8

1.0 0.8 0.6 0.4 0.2 0.0 200

250

300

350

Wavelength (nm)

Figure 5. The UV–Vis absorption spectra of pepsin (A), α-amylase (B) and trypsin (C) incubated with various concentrations of PS-NPs. NPs concentrations from b to g: 0, 0.01, 0.03, 0.05, 0.07, and 0.1 mg/ml, respectively. a: bare PS-NPs, 0.1 mg/ml.


350


30

A

a

40

f

30

Page 36 of 43

B a

20 Intensity

Intensity

f

10

20

10

0

0 300

330

360

390

420

300

Wavelength (nm)

100

330

360

390

wavelength (nm)

C a

Intensity

f 50

0 300

330

360

390

420

Wavelength（ nm（

Figure 6. The fluorescence emission spectra obtained for pepsin (A), α-amylase (B) and trypsin (C) incubated with various concentrations of PS-NPs. NPs concentrations from a to f: 0, 0.01, 0.03, 0.05, 0.07, and 0.1 mg/ml.


420

Page 37 of 43


A 5

Δλ =15 nm

16

a

4

12

f

3

Intensity

Intensity

20

8

4

Δλ= 60 nm

a f

2

1

0 240

280

0 240

320

280

Wavelength (nm)

320

Wavelength (nm)

B 6

30 Δλ = 15 nm 25

a

a

20

f

4

f Intensity

Intensity

Δλ=60 nm

15

2

10 5 0 240

280

320

Wavelength (nm)


0 240

280 Wavelength (nm)

320


Page 38 of 43

C 70

16 Δλ = 15 nm

60

a

a

12

50

f

10

f 40

Intensity

Intensity

Δλ = 60 nm

14

30 20

8 6 4

10

2

0 240

280

320

Wavelength (nm)

0 240

280 Wavelength (nm)

Figure 7. Synchronous fluorescence spectra of pepsin (A), α-amylase (B) and trypsin (C) in the presence of different concentrations of PS-NPs. NPs, a - f: 0, 0.01, 0.03, 0.05, 0.07, and 0.1 mg/ml.


320

Page 39 of 43


A

1.6

Pepsin 298K, R2 = 0.9742 304k, R2 = 0.9893

F0/F

1.4

310K, R2 = 0.9685 304K

1.2

1

0.8 0

1

2

3 4 -4 [Q] (10 mol/L)

6

298K, R2 = 0.99

α-amylase

B

1.8

5

304k, R2 = 0.9649 310K, R2 = 0.9576 304K

1.6

F0/F

1.4 1.2 1 0.8 0

1

2

3

4

5

[Q] (10-4 mol/L)


6


Page 40 of 43

C

1.8

298K, R2 = 0.9951

Trypsin

304k, R2 = 0.9984

1.6

310K, R2 = 0.9867 304K

F0/F

1.4 1.2 1 0.8 0

1

2

3

4

5

[Q] (10-4 mol/L) Figure 8. The Stern–Volmer curves of pepsin (A), α-amylase (B) and trypsin (C) quenched by NPs at 298, 304, and 310 K. The error bar corresponds to the standard deviation for n = 3.


Page 41 of 43


6.0

A

lnKa

5.9 5.8 5.7 5.6

2

R = 0.9938

5.5

0.00324

0.00328

0.00332

0.00336

1/T (K-1)

6.1

B

6.0

lnka

5.9 5.8 5.7

2

R = 0.9949

5.6 0.00324

0.00328

0.00332

0.00336

1/T (K-1)

8.0

C

7.5

lnka

7.0 6.5 6.0 5.5 2

R =0.9994

5.0 0.00324

0.00328

0.00332

0.00336

1/T (K-1)



Figure 9. Van’t Hoff plots of PS-NPs interaction with pepsin (A), α-amylase (B) and trypsin (C).


Page 42 of 43

Page 43 of 43


Graphic Abstract

Pepsin

Protein Corona

Polystyrene nanoparticles

α-amylase

Trypsin


Hard Corona

Formation of protein corona on nanoparticles with digestive enzymes

Recommend Documents