Formation of Protein Corona on Nanoparticles with Digestive Enzymes

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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

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Journal of Agricultural and Food Chemistry

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Formation of protein corona on nanoparticles with digestive enzymes

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in simulated gastrointestinal fluids

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

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

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Shandong Province, 266109, China)

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

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[email protected])

ACS Paragon Plus Environment


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

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

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

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

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

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

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

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

16

and ultraviolet visible absorption spectra suggested the interaction between the

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nanoparticles and the enzymes occurred. Synchronous fluorescence spectra showed

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

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The thermodynamic parameters suggested that the interaction was mainly driven by

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the hydrogen bonds and van der Waals forces.

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

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fluids


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INTRODUCTION

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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delivery capability of AuNPs and dictating the molecular targeting and biodistribution

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

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

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

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

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exploitation of the interaction between NPs and protein.12



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

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

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

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behavior of NPs and their interactions with biological molecules in gastrointestinal

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

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

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

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

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

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

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

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

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

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hydrodynamic diameter of zinc oxide nanoparticles remarkably increased because of

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

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

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

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

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

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

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

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

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

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

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


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

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morphology around the NPs was visualized using transmission electron microscopy

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

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

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

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

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

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

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

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

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

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MATERIALS AND METHODS

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

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

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

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

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

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

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

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

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

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

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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



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

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

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

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

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

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

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solution

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

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

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

to

remove

unbound

proteins,

obtaining

pepsin-coated

PS-NPs,

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Transmission Electron Microscopy. To directly confirm the adsorption of

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

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

110

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

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

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

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

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observed with TEM.

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

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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.


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

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

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

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

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

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

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

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

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

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detected at 562 nm.

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

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

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

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

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

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

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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

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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.

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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,

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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.

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

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

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

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

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

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

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5% (p < 0.05).

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RESULTS AND DISCUSSION

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

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

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

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

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

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

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

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

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

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

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

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

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


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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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three times.



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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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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

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

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of up to 100 μg/mg.

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Investigation of protein–particle interactions


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

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

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

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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

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

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

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

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

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

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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

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

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

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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.

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

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

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

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

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

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

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

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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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backbone and reduced the hydrophobicity of the microenvironment of trypsin.

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

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

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

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

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

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when the concentration of cellulose nanocrystals gradually increased, thus suggesting


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the interaction between cellulose nanocrystals and α-amylase.

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

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

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

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

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the particle concentration increased, indicating the quenching of protein fluorescence

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

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

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

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

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

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

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

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

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the occurrence of the interaction between HSA and TiO2 NPs.

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Synchronous Fluorescence Spectra. The variation in the synchronous fluorescence

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spectra can reflect the change in the microenvironment surrounding tyrosine or

286

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

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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

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

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

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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



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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.

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Fluorescence Quenching Mechanism. To further elucidate the fluorescence

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

308

process the quenching data35:

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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)

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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


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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

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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

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