Exploring the Heterogeneity of Nanoparticles in Their Interactions with

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Article

Exploring the Heterogeneity of Nanoparticles in Their Interactions with Plasma Coagulation Factor XII Fang Hao, Qian S. Liu, Xi Chen, Xingchen Zhao, Qunfang Zhou, Chunyang Liao, and Guibin Jiang ACS Nano, Just Accepted Manuscript • Publication Date (Web): 11 Feb 2019 Downloaded from http://pubs.acs.org on February 11, 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 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Exploring the Heterogeneity of Nanoparticles in Their Interactions with Plasma Coagulation Factor XII

Fang Hao,a,b Qian S. Liu,a Xi Chen,c Xingchen Zhao,a Qunfang Zhou,a,b,d* Chunyang Liao,a Guibin Jianga,b

a

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for

Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China b

College of Resources and Environment, University of Chinese Academy of Sciences, Beijing

100049, China c Waters

Corporation, Asia Pacific Headquarter, Shanghai, 201206, China

d Institute

of Environment and Health, Jianghan University, Wuhan, 430056, China

* Correspondence to: Dr. Qunfang Zhou, State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China. E-mail: [email protected] 1

ACS Paragon Plus Environment

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Tuning the characteristics of nanoparticles (NPs) would be promising in improving their biocompatibilities, regarding biosafety and nanodrug considerations. Due to the high priority of the artificial NPs in contacting the circulatory system, understanding their interactions with plasma zymogens is of great importance. Four kinds of NPs, including 5 nm gold NPs (GNP-5), 5 nm and 20 nm silver NPs (SNP-5, SNP-20), and 20 nm silica NPs (SiNP-20), were investigated for their interactions with the coagulation factor XII (FXII). GNP-5 adsorbed FXII in a standing-up mode, and exhibited high binding affinity for the heavy chain of the protein without altering its secondary structure or inducing its activation. In contrast to GNP-5, FXII adsorption on the other tested NPs was in lying-down mode, and their interactions with FXII induced its conformational changes, thus causing the evident zymogen cleavage. The structural alterations and activation of FXII induced by the NPs exhibited in specific surface area dependent manners, which were related with different NP cores and sizes. Additionally, the enzymatic activity of -FXIIa was also influenced by NP incubation, and the alterations were dependent on the specific characters of the NPs as evidenced by the enzymatic inhibition effect of GNP-5 (noncompetitive) and SNP-5 (competitive), and enhanced enzymatic catalysis abilities of SNP-20 and SiNP-20. The interesting findings on the heterogeneity of NPs in their interactions with plasma FXII not only revealed the underlying mechanism for NP-triggered hematological responses, but also suggested the crucial role of tuning NP parameters in their potential bioapplication, like nanodrug design.

2


Page 2 of 51

Page 3 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

KEYWORDS:

nanoparticles

(NPs),

coagulation

conformational change, enzymatic activity

3


factor

XII,

heterogeneity,

ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Nanoparticles (NPs) possess excellent physicochemical and biological properties, which are greatly distinctive from bulk forms. The extensive usages of NPs have been explored in diverse aspects, including medical area like drug carrier platform, clinical imaging, and therapy agent etc.1-4 Nevertheless, the increasing development of NPs concomitantly bring up potential exposure risks to the environment and human bodies, thus causing the unintended harmful effects.5,6 Once NPs enter into bodies, they, in most instances, would firstly circulate in vascular system, adsorb and interact with a wide range of plasma proteins to form NP-based protein corona,7 which crucially determines the bioactivities of NPs.8 In other words, the contact of plasma proteins with NPs may physiologically alter their conformational structures and biological functions.9-11 Therefore, the haemocompatibility testing, based on the interaction between NPs and plasma proteins, is one of the most important aspects for nanosafety evaluation, which critically guides the design of effective nanomedicine as well.12 The corona composition of NPs may evolve during the conditioning in aqueous environment, wherein, part of proteins in corona, also known as hard corona, remain constantly for a long time on NP surface,13 while some other proteins, i.e. soft corona, exchange dynamically with those in ambient solution. The hard corona, instead of the soft corona, determines the biological fates of NPs in body fluids, such as plasma and cytoplasma,14 which is thus considered as the finger-print of NPs.15 Previous studies have revealed that the coagulation cascade proteins usually exist in the hard corona of NPs,15-17 and the members in kallikrein-kinin system (KKS), including Hageman factor (FXII), prekallikrein (PPK), and high-molecular-weight kininogen (HK), can be 4


Page 4 of 51

Page 5 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

evidently enriched therein.16 Given the cascade activation of the KKS is classically initiated by the auto-activation of FXII when contacting the negatively charged surface, and many artificial NPs with negative surface charges are potential FXII activators,18 these proteins would definitely be involved in NP-mediated bioactivities, such as vascular permeability, inflammation, the intrinsic coagulation etc.18-21 The activity screening using FXII activation would thus be crucial in explaining haemocompatibilities of the NPs. Multiple physicochemical factors of NPs, such as size,22 shape,23 coating,24 and sedimentation25 would influence the corona formation, due to the varying binding affinities for proteins.26 The heterogeneity of NPs based on the single characteristics is now being hotly debated, and extensively explored, regarding particle size, shape, coating etc. For instance, Setyawati et al.27 studied the impact of particle size on gold NPs induced endothelial leakiness, and confirmed that those with the size of 20 nm, instead of 10 or 50 nm, were prone to disturbing endothelial cell junctions. The physical forces, like shear force, would influence the biological behaviors of NPs when they enter into blood circulation. Tang et al.28 investigated the vascular distribution of NPs with different densities, and found heavier NPs were apt to flow along margin under blood shear force. Conclusively, the heterogeneity of NPs would influence their hematological behaviors. As one of the most important zymogen system in blood coagulation process,29 how the FXII activation is influenced by the heterogeneity of NPs is of high importance in explaining their hematotoxicity, and would be very helpful in guiding the design of highly secured nanomaterials. 5


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

In this work, we tested the adsorption and activation of FXII induced by a series of NPs, including gold, silver and silica NPs with different particle sizes (i.e. GNP-5, SNP-5, SNP-20 and SiNP-20), and the effects were compared based on their specific surface areas. The binding geometric characteristics between FXII and NPs were explored by dynamic light scattering (DLS) and theoretical calculation. The binding affinity parameters were obtained from microscale thermophoresis analysis (MST). The binding sites and heterogeneity of NPs in inducing FXII activation were revealed by hydrogen/deuterium exchange mass spectrometry (HDX-MS). The conformational change and auto-activation of FXII upon the treatments of NPs were characterized by photometric analysis and Western blot. The findings revealed the distinct FXII adsorption and activation regulated by the NPs with specific characteristics, and testing the heterogeneity of the NPs in activating plasma zymogens would be promising in designing nanomaterials for specific medical purposes.

6


Page 6 of 51

Page 7 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

RESULTS AND DISCUSSION The Characteristics of NP Matrices. The intrinsic properties of NPs, like core density,30 surface coating,31 and particle size27 have been demonstrated to exert vital effects on their biological activities, and the specific surface area of NPs is also a crucial regulatory factor in nanotoxicology. To characterize how NPs with different cores, particle sizes and specific surface areas influenced their interactions with FXII, four types of NPs, including gold NPs with the particle size of 5 nm (GNP-5), silver NPs with the particle sizes of 5 nm and 20 nm, respectively (SNP-5, and SNP-20), and silica NPs with particle size of 20 nm (SiNP-20), were chosen and characterized in Figure S1. As the images of transmission electronic microscopy (TEM) in Figure S1A showed, all nanoparticles were spherical with good dispersion. The dry size distribution based on the calculation of more than 100 particles per type, was fitted with Gauss function, and showed the mean particle sizes of 5.1 ± 0.6 nm, 5.2 ± 0.9 nm, 18.5 ± 2.4 nm, and 21.4 ± 2.7 nm for GNP-5, SNP-5, SNP-20 and SiNP-20, respectively. Their hydrodynamic sizes were 5.59 ± 1.11 nm, 5.92 ± 0.48 nm, 25.58 ± 2.80 nm, and 21.57 ± 1.57 nm (Figure S1B), which were well consistent with the results of their dry sizes. The zeta potentials were all negative, ranging from –18.10 mV to –49.00 mV (Figure S1B). As for localized surface plasmon resonance (LSPR) absorption spectra located in visible wavelength zone obtained from NIR-3600 (Shimadzu, Japan) with a quartz cell (Figure S1C), three kinds of NPs, including GNP-5, SNP-5 and SNP-20, showed the characteristic maximum absorbance values at 520 nm, 396 nm and 406 nm, respectively, while SiNP-20 had no detectable 7


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

absorbance in the range of 300-500 nm. The Adsorption Geometry of FXII on the NPs. Once FXII is exposed to the NPs, its adsorption to the surface of NPs was the first step, and the study on the adsorption behavior would help to explain the interaction between the protein and NPs. Based on the measurements of the hydrodynamic sizes of NPs and FXII-NP complex, the size difference was evaluated, and the results in Figure 1A showed the hydrodynamic diameter increases of FXII-NP complexes were 7.16-10.82 nm, whose sizes were significantly larger than those of the corresponding NPs (p < 0.05 or 0.01). The adsorption of FXII molecule on the NPs determines their hydrodynamic size changes, and the adsorption density, orientation and deflection of FXII on NPs were crucial parameters in evaluating this process. The hydrodynamic size of FXII (i.e. 2RFXII) measured by DLS was 7.50 nm (Figure S2), which was consistent with reference value.32 Using the hydrodynamic sizes of the NPs, FXII and the FXII-NP complex, the maximum numbers of FXII adsorbed on the tested NPs (Nmax) were calculated in this study, according to the method described by Mattoussi et al.33 The results shown in Figure 1B indicated that Nmax of GNP-5, SNP-5, SNP-20, and SiNP-20 were 6.76 ± 1.72, 3.25 ± 1.34, 29.38 ± 1.69, and 23.42 ± 3.30, respectively, and the protein densities on each NP calculated by their dry surface areas (DFXII) were 0.083, 0.038, 0.027 and 0.016 nm-2 for GNP-5, SNP-5, SNP-20, and SiNP-20, separately. Apparently, GNP-5 adsorbed more FXII than did SNP-5, and the adsorption capability of SiNP-20 for FXII was lower than that of SNP-20. This suggested the binding affinity for adsorbed FXII was highly 8


Page 8 of 51

Page 9 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

dependent on NP composition,34 which was consistent with previous findings.35 Regarding the surface curvature of the NPs, the surface adsorption density of FXII decreased from 0.038 to 0.027 nm-2 for SNP when the surface curvature decreased from 0.38 nm-1 (SNP-5) to 0.10 nm-1 (SNP-20). It was reasonable that FXII would face lower steric hindrance when confronting the particulate surface with higher curvature. Considering the hydrodynamic diameter changes (ΔD) of 7.16-10.82 nm introduced by FXII adsorption on the NPs and the hydrodynamic diameter of 7.50 nm for FXII itself, a monolayer, instead of a multilayer, of FXII was suggested to adsorb on the tested NP surfaces. This assumption of the monolayer adsorption mode was consistent with previous investigations on the geometry of NP-protein aggregates, like studies on the interaction of BSA with silica NPs,36 and the adsorption of HSA to quantum dots.37 According to the shape of FXII revealed by the atomic force microscopy (AFM) in Figure S3, the molecular appeared in elongated shape with major and minor axes. The calculation of the semi-axis ratios of FXII based on half of the ΔD values and the radii of the circular footprints from protein projections on NP surfaces (Table S1) indicated different adsorption geometries of FXII on NP surfaces (Figure 1C). More specifically, the adsorption orientation of FXII on GNP-5 was longitudinal (i.e. standing-up mode), and those on SNP-5, SiNP-20 and SNP-20 were latitudinal (i.e. lying-down mode) due to the higher semi-axis ratio of FXII on GNP-5 (1.24) than those on the other tested NPs (0.39-0.53). Similar finding on the adsorption orientation of the protein to the NPs was previously reported by Huang et 9


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

al.,31 who revealed that BSA attached onto the surface of MUS/brOT-coated GNPs with its triangular face, but adsorbed on MUS/OT-coated GNPs with its vertex of the longest dimension. Further evidences from 20 nm gold NPs (GNP-20, Figure S4) showed the similar adsorption density and orientation of FXII on GNPs, suggesting core materials of the NPs determined the protein adsorption geometries. The deflection of FXII on NP surface shown in Figure 1C, described the angle between the links of NP circular center with both ends of the protein. According to the calculation method suggested by Hill et al.,38 the results in Figure 1B showed that GNP-5 had smaller deflection angle (89.03ο) than did SNP-5 (133.85ο) because of the tight longitudinal adsorption of FXII on GNP-5, while SNP-20 and SiNP-20 had similar deflection angles ( 42.31ο and 47.53ο), revealing the adsorption mode of FXII on their surfaces were the same. When the deflection angles of FXII on the NPs with different particle sizes were compared, it was found that SNP-20 with low curvature had much smaller deflection angle than did SNP-5. As illustrated in Figure 1C, this difference was caused by the distinct particle radii of these two kinds of NPs, the adsorption geometry (lying-down mode) of FXII on their surfaces were the same though. The Binding Affinities of FXII for the NPs. The binding affinities of proteins for the NPs determine the composition of the corona,26 and the characters of the corona mediate the biological fate of the NPs.15,39 To understand the interaction of FXII with the tested NPs during their protein corona formation, MST was used to evaluate the binding affinities of FXII for GNP-5, SNP-5, SNP-20 and SiNP-20, 10


Page 10 of 51

Page 11 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

respectively, as any change of the fluorescently labelled FXII due to its binding with the NPs would alter its movement along temperature gradient. The results in Figure 2A showed the representative thermophoresis curves of GNP-5 at the concentrations of 0.0054-88.12 pM, revealing that the time traces of FXII shifted along with its binding to GNPs in titration concentration-dependent manner. Similar results were observed for SNP-5, SNP-20 and SiNP-20, as shown by the typical thermophoretic depletion profiles depicted in Figure S5, indicating the binding of FXII with the tested NPs. The sigmoidal binding curves in Figure 2B indicated FXII exhibited different binding affinities for four tested NPs, wherein, the highest unbound concentrations (HUC) of the NPs were 0.013, 0.37, 7.81, and 22.03 pM for SNP-20, SiNP-20, SNP-5 and GNP-5, respectively. This could be correlated with the number of FXII adsorbed the NPs (Figure 1B). The more FXII molecules adsorbed on the NPs, the less the NPs were needed to influence the thermophoretic depletion profile of the probed FXII. The contradictory order for GNP-5 and SNP-5, regarding their Nmax and HUC values (Figure 1B and 2B), could be contributed by the different binding modes of FXII as depicted in Figure 1C. Additionally, the thermophoretic amplitudes, due to the contributions from multiple factors of the bound complex, including the size, charge and structure,40 were determined. Apparently, the thermophoretic amplitudes of GNP-5 (0.82) and SNP-5 (1.10) were much higher than those of SNP-20 (0.40) and SiNP-20 (0.42) within the tested titration concentration ranges (Figure 2B), suggesting the distinct physical characteristics of the formed bioconjugates of NP-FXII herein. 11


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

According to the thermophoretic depletion profiles of the tested NPs, the parameters, including the dissociation constant (Kd), the adsorption constant (Ka) and Gibbs free energy (ΔG) were calculated, and the results were listed in Figure 2C. The Kd values ranged from 0.25 nM to 8.24 nM, which were the reciprocals of Ka values. When the NPs grouped by the particle size were compared, FXII exhibited relatively higher binding affinities for GNP-5 and SNP-20, which were consistent with their Nmax results (Figure 1B). The negative values of ΔG indicated that the binding of FXII with the NPs was an exothermic process, wherein, GNP-5 and SNP-20 had the relatively lower ΔG values when compared with the other two kinds of NPs, which was consistent with their Ka results. The Interaction Domains of FXII for the NPs. Considering the distinct binding modes and binding affinities of FXII for the tested NPs observed above, the study on the specific binding sites of NPs on FXII would be helpful to reveal their interactions. Using HDX-MS, the detailed binding sites and the potential structure changes, were thus explored during the interaction process of FXII with the NPs. Based on the workflow of H/D exchange process shown in Figure 3A, the deuteration of FXII in D2O with the presence of NPs was compared with that of the protein in the absence of NPs, and the representative isotopic peaks of different peptides with or without four types of NP treatments were shown in Figure 3B. For example, the binding of GNP-5 with the amino acid sequence (20-40) partially blocked its centroid mass peak shift from the m/z value of original peptide in H2O (m/z, 626.34) to that of its deuteration form in D2O (m/z, 627.85), resulting in the corresponding m/z of 12


Page 12 of 51

Page 13 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

626.84. The centroid mass of peptide 378-389 in FXII with SNP-5 treatment (m/z, 621.20) was higher than that of the corresponding peptide deuterated alone in D2O (m/z, 620.84), indicating SNP-5 induced the conformational alteration of the peptide favorable for the faster deuteration. As for SNP-20 treatment, it rarely altered the centroid mass of peptide 371-377 (m/z 703.94) when compared with the corresponding peptide isotope peaks from deuterated FXII without NP incubation (m/z, 703.77), showing that SNP-20 did not bind with this peptide region of FXII or induce its structural change. Similar interaction mode was observed for SiNP-20 in peptide 390-396, whose centroid mass in the presence of SiNP-20 was similar to that in the absence of the NPs during deuteration (m/z, 456.02 vs 456.03). Quantitative analysis of the relative deuterium exchange rate (D%) in Table S2 indicated that the values of GNP-5, SNP-5, SNP-20 and SiNP-20 were in the ranges of 33-100%, 70-151%, 81-137% and 71-128%, respectively. Based on the color mapping of the D% values in different treatments, the interaction modes of the tested NPs were comparatively depicted in Figure 3C. It was clear that GNP-5 could bind tightly with FXII in several specific domains, like sequence 20-40 (peptide I), 205-224 and 225-233 (peptide III) with the D% values less than 50%, which belonged to the negatively charged surface binding area located near N-terminal of the protein. No conformational changes were induced by GNP-5 for all peptides detected (peptide I-VII). In contrast to GNP-5, the relatively weak binding of SNP-5 only occurred in the amino acid regions of 205-224 and 281-287 (D% of 73% and 70%). The treatment of SNP-5 also caused the accelerated H/D exchange in peptide 254-264 (IV), 371-377 13


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(VI) and 378-389 (VII) of FXII (D% of 135%, 129%, and 151%, respectively), due to the loss in hydrogen bond integrity from the secondary structure destruction of the protein.41 Regarding SNP-20 and SiNP-20, they showed very similar interaction modes for different domains of FXII, dominated by the induction of conformational changes in the light chain of FXII. The hierarchical clustering analysis for deuteration data of the peptides in different treatments confirmed that the interaction of GNP-5 with FXII was different from those of the other tested NPs, while the interactions of SNP-20 and SiNP-20 were the most alike (Figure 3C). The different interaction modes of the NPs for FXII would consequently cause the distinct biological characteristics of the protein. Conformational Changes of FXII upon NP Interaction. The interaction of the protein with the NPs would result in its conformational changes, which can be mediated by the properties of the NPs, like particle sizes and surface coatings etc.42,43 The aromatic fluorophores, like tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) in the protein are sensitive to the microenvironment, and the NP-induced protein conformational change usually causes the changes in its fluorescence characteristics, like fluorescent quenching44 or enhancement,45 due to the different accessibility of nanomaterials to protein fluorophores.46,47 The above HDX-MS data suggested that four tested NPs could bind with or induce structural changes of FXII. To further confirm this result, the conformation of FXII was characterized by measuring the fluorescence of the protein with or without NP treatments, based on the spectroscopic characteristics of FXII.48 The representative profiles for the emission 14


Page 14 of 51

Page 15 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

spectroscopic curves in the range of 290-450 nm were depicted in Figure 4A, which showed that NP treatments mainly exerted fluorescent quenching effects on FXII. The analysis of specific surface area-related effects on the fluorescence intensity at 350 nm revealed different change profiles for the tested NPs (Figure 4B). The fluorescence quenching of FXII was positively correlated with the specific area of GNP-5 treatment, and the linear plot was well fitted by the Stern-Volmer equation after the elimination of inner filter effect (IFE) (Figure S6) with R2 of 0.98 (Table S3). The biomolecular quenching constant (kq) of 8.0×1016 M-1S-1 in Table S3 was much higher than the maximum diffusion collision constant (2.0×1010 M-1S-1),49 suggesting the high quenching ability of GNP-5, and its quenching mode was dominated by static one.50 Conclusively, the fluorescence of FXII was quenched by the specific perturbation of GNP-5 in the microenvironment of Trp and Tyr. As for SNP-5, SNP-20 and SiNP-20, the protein fluorescence intensities decreased, then increased with the increases of NP specific surface areas, exhibiting the U-shaped responses (Figure 4B). The fluorescence quenching was believed to be ascribed to the microenvironment change of fluorophores, i.e. the transformation from a hydrophobic surrounding to a hydrophilic one. The spherical SNPs have been reported for their abilities in quenching protein fluorescence,51 which was briefly consistent with the findings herein. Nevertheless, with the increase in the specific surface areas of SNP-5 and SNP-20, their surface plasmon resonances were induced as long as the distances of NP surfaces to the fluorophores were close enough.52 Therefore, the backbone of FXII got unfolded and the cleavage was induced (to be described below). The 15


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

increasing exposure of the fluorophores to silver surface plasmon consequently enhanced the fluorescence. Similar finding was also reported for gold nanorods, which was attributed by coupling with longitudinal plasmon resonance.53 In contrast to SNPs, the incubation of SiNP-20 at relatively low specific surface area (3, 10 and 20 m2/g) loosed the backbone of FXII, causing the decrease of the protein fluorescence intensity, but at relatively high specific surface area (30, 40 and 60 m2/g), the fluorescence intensities surprisingly increased. This result suggested that SiNPs at high specific surface areas could not accelerate the conformational change of FXII. Instead, the microenvironment integrity of the protein fluorophore recovered somehow. Therefore, the U-shaped fluorescence alteration in FXII suggested the complicated transformation of the fluorophore microenvironment due to the incubation of SiNPs at different specific areas. The circular dichroism (CD) spectrum, as a commonly used technique for the characterization of protein conformation, was further used to test the alterations in the secondary structure of FXII in the presence or absence of the NPs. The results in Figure 4C showed that the treatment of GNP-5 at 10 and 60 m2/g caused little changes in the percentages of α-helix, β-sheet, β-turn or random coil of FXII, whose secondary contents were similar to the results in previous work,54 and the variations were less than 3.89%, suggesting the secondary structure of FXII was intact. As for the incubation of SNP-5 and SNP-20, the contents of α-helix and random coil in FXII decreased with the increase of NP specific surface areas, while that of β-sheet showed the opposite trends (Figure 4C), revealing the disintegration in the secondary structure 16


Page 16 of 51

Page 17 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

of FXII and the extension of the peptide backbone.47,55 Regarding SiNP-20, its treatment at 10 m2/g caused a decrease of α-helix in FXII to 3.13%, and an increase of β-sheet to 48.12%, while that at 60 m2/g recovered the contents of α-helix and β-sheet to 7.07% and 43.00%, respectively (Figure 4C). This phenomenon could be related with the compromised FXII cleavage at 60 m2/g SiNP-20 treatment (to be described below). FXII Activation Regulated by the NPs. The adsorption of FXII on the negatively charged surface caused its conformational change and the subsequent autoactivation, as evidenced by the cleavage of the zymogen FXII (~75 kDa) and the formation of the dominant heavy chain of FXII at 50 kDa (FXIIa).18,56 Understanding how different adsorption geometries of FXII on the NPs influence its activation would be helpful in explaining the potential biological functions of the NPs. The immunoblotting assay for the purified FXII incubated with different NP matrices showed that the treatment of GNP-5 caused no alterations in FXII electrophoresis behavior (Figure 5A), revealing no influence was induced by GNP-5 on FXII activation. Distinct from GNP-5, the other three kinds of NPs, including SNP-5, SNP-20 and SiNP-20, induced apparent cleavage of FXII, and the decreases in FXII zymogen band densities and increases in activated FXII band densities exhibited in the specific area dependent manners (Figure 5A). The difference between GNP-5 and the other NPs in causing FXII cleavage could be closely related with the adsorption geometry, wherein, the activation site of FXII was conserved in standing-up mode on GNP-5, whereas, the lying-down mode of the protein could result in exposing more 17


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

contacting interface, including the activation site, to the negatively charged surfaces of the other three NPs. Moreover, the analysis of the relationship between FXII cleavage and the specific areas of the NPs showed different response curves for the tested NPs, i.e. S-shaped curve for SNP-5, positive linear fitting curve for SNP-20, and inverted U-shaped curve for SiNP-20, respectively (Figure S7). To further evaluate the biological significance of NP-induced FXII activation, a proof-of concept experiment was performed using human blood samples. The results in Figure S8 showed that GNP-5 did not cause FXII cleavage in human plasma, while the other tested NPs (i.e. SNP-5, SNP-20, and SiNP-20) induced FXII cleavage in specific surface area dependent manners, as evidenced by the formation of FXIIa (i.e. 50 kDa band). These findings were consistent with the results from in vitro assays (Figure 5A), confirming the potential hematological effects of the NPs by interacting with the coagulation factor of FXII observed from in vitro assays. The activation of FXII induced by the NPs was also evaluated using the substrate method (Figure S9), and FXIIa enzyme activity change in each treatment was depicted in Figure 5B. Apparently, GNP-5 incubation had no effect on the activation of FXII. On the contrary, the treatments of SNP-5, SNP-20 and SiNP-20 showed obvious induction in FXII activation, and the correlation analysis for the specific areas of the NPs and FXIIa activity increases showed the S-shaped, positive linear fitting, and inverted U-shaped curves, respectively (Figure 5B). This result was well consistent with the finding of FXII cleavage from immunoblotting assay (Figure 5A, S7). 18


Page 18 of 51

Page 19 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Considering the bound protein on NP surface and the free one in the solution could have different profiles, the “pull-down” assay was performed according to the protocol shown in Figure 5C. The results in Figure 5D showed the free FXII in the supernatant decreased with the increase of GNP-5 specific area, while the bound FXII in the pellet showed the opposite trend, which indicated the efficient adsorption of the protein without activation on GNP-5 with high specific surface areas. As for SNP-5 and SNP-20 groups, the decreasing trends of FXII were observed in the supernatants, and the increasing trends of FXII and FXIIa were detected in the pellets when related to the specific surface areas of the NPs. No FXIIa of 50 kDa was detected in the supernatants, showing FXII activation was triggered on SNP surfaces, and the active form of the protein could closely bind to SNPs. In view of SiNP-20, the different profiles for both FXII and FXIIa exhibited in the supernatant and the pellet as evidenced by Figure 5D. That was, decreased FXII with low level of FXIIa appeared in the supernatant of 10 m2/g treatment group, and all protein vanished in supernatants of the other treatments (20-60 m2/g). Correspondingly, FXII levels in the pellets increased with the specific surface area of SiNPs, while decreasing trend was observed for FXIIa level therein. Apparently, this activation mode of FXII on SiNPs was consistent with the results from Figure 5A and 5B. The different capabilities of the NPs in inducing FXII activation was strictly regulated by the particle core chemistry and the interaction mode-related conformational changes of the protein. The compromised FXII cleavage and FXIIa activity observed in SiNP-20 with high specific surface areas (40 and 60 m2/g) were potentially regulated by the altered 19


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

interaction mode of SiNP-20 in regulating the secondary structure of FXII as characterized by spectroscopic analysis (Figure 4B and 4C). Considering different cleavage sites in FXII chain,57-59 the enhanced chemiluminescence detection based on Western blot was performed to analyze the formation of cleaved FXII bands at trace levels, so that how the NPs induced FXII cleavage could be revealed. The results in Figure S10A showed no band in the range of 15-50 kDa appeared in any of GNP-5 treatments, confirming no cleavage was induced by GNP-5. As for SNP-5 and SNP-20 groups, besides the formation of the dominant 50 kD band, another protein band, i.e. 28 kDa, came into being upon the incubation of the NPs at relatively high specific areas (10-60 m2/g for SNP-5, and 10-30 m2/g for SNP-20, respectively (Figure S10A). Regarding SiNP-20, 5 cleaved bands, including 50, 40, 30, 28, and 12 kDa, formed in 10-60 m2/g treatment groups, showing more complex cleavage behavior of FXII when compared with those in SNP treatments. The activation of FXII zymogen (Figure S10B), as a single-chain polypeptide of 596 amino acid residues, was reported to experience conformational change upon the interaction with negatively charged surfaces, and proteolytic cleavage at amino acid residues Arg353-Val354 (Site 1) via autoactivation or more efficient catalysis by plasma kallikrein (PK), thus forming a two-chain protein (i.e. a heavy chain of 52 kDa with 352 residues and a light chain of 28 kDa with 244 residues) connected by the Cys340-Cys467 disulfide bond (i.e. α-FXIIa).54,57,58 The heavy chain of α-FXIIa was further cleaved at Arg 343 (Site 2) and Arg334 C termini (Site 3) by PK, thus yielding the 2 polypeptide chains including 2 kDa heavy chain 20


Page 20 of 51

Page 21 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

remnant and a 28 kDa catalytic domain covalently held together by the same disulfide bond.58 Reduction of the disulfide bond liberated the light chain of -FXIIa (28 kDa) with the catalytic domain.59 Additionally, Site 4 at Arg264 in the heavy chain was another potential cleavage point suggested previously.60,61 All theoretically possible fragments thus included 12, 28, 30, 40, and 50 kDa (Figure S10B), due to FXII cleavage at 4 different sites. The diverse fragmentation formation modes in Figure S10A and S10B indicated SNPs (SNP-5 and SNP-20) and SiNP-20 could induce different cuttings in FXII chain under PK-free condition in vitro, due to their distinct interactions with the protein. Enzyme Activity Changes in -FXIIa upon the bioconjugation of the NPs. As the activated FXII fragment (50 kDa) was found to aggregate on the surface of the NPs (Figure 5D), how its enzymatic activity was influenced would help to further elucidate the effects of the NPs. Based on the incubation of the NPs with the purified human -FXIIa (characterized by 50 and 30 kDa bands, Figure S11), the changes in the enzyme kinetics were analyzed by evaluating the parameters, like Vmax, Km, kcat, and Ceff.32 The results in Figure 6A showed that the treatments of GNP-5 and SNP-5 decreased the initial velocity of the enzyme reaction (V0) in the specific surface area-related manners, while the incubation of SiNP-20 and SNP-20 showed the opposite effects, i.e. V0 was elevated with the increase of NP specific areas. The calculation of the enzyme kinetic parameters for -FXIIa in different treatments showed that Vmax of the enzyme with GNP-5 treatment decreased with the increase of NP specific area. As for SNP-5 treatment, Vmax of -FXIIa showed little 21


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

change. Both SNP-20 and SiNP-20 treatments greatly increased Vmax, indicating their positive effects on the enzymatic activity of -FXIIa, which were consistent with V0 changes. Michaelis-Menten constant, denoted by Km, reflects the binding affinity of enzyme for its substrate. The smaller value of Km indicated the stronger binding affinity of the enzyme with the substrate. In GNP-5 treatments, Km values remained almost unchanged (around 156 μM, Figure 6B), showing GNP-5 did not influence the binding affinity of the enzyme for the substrate. The treatments of SNP-5 and SiNP-20 increased Km values, revealing that the binding affinity between -FXIIa and substrate was compromised. The variations in Km value of -FXIIa induced by SNP-20 indicated the complex interaction mode for this kind of NPs at different specific areas. To further elucidate the effect of NPs on the catalytic efficiency of -FXIIa, the catalysis constant (kcat) and the catalysis efficiency (Ceff) were evaluated for different treatments. The kcat and Ceff values of -FXIIa decreased in both GNP-5 and SNP-5 treatments (Table S4), showing their inhibition effects on the enzyme. In contrast, SNP-20 increased these two parameters, and SiNP-20 increased kcat, but had little effect on Ceff. (Table S4). This indicated that these two NPs could enhance the catalysis reaction of -FXIIa, while exerted distinct effects on its catalysis efficiency. Regarding the interaction modes of the tested NPs on -FXIIa (Figure S12), GNP-5 induced noncompetitive inhibition effect on this enzyme, as it could inhibit the dissociation of enzyme-substrate complex, thus decreasing Vmax, but not Km. In contrast to GNP-5, SNP-5 caused increase of Km, but not Vmax, suggesting competitive 22


Page 22 of 51

Page 23 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

inhibition was induced by its direct binding with the catalytic site of the enzyme or the nearby domain, resulting in the turnover of the enzymatic reaction from steric hindrance.62 As for the NPs with relative large particle size (i.e. SNP-20 and SiNP-20), their positive effects on enzyme activity, as evidenced by the increased Vmax and kcat values (Figure 6B, Table S4), suggested -FXIIa adsorbed on plane surface remained functional domain available, and the catalysis could be enhanced by the favorable conformational changes of the enzyme upon NP treatments. The findings above also suggested that the relatively low curvature of the NPs could have vital impact on improving the enzyme activity.63 In addition, the negatively charged surface of the NPs could induce ordered adsorption of FXII, which facilitated the catalytic reaction of -FXIIa as well.64,65 Altogether, the distinct effects induced by different NPs on -FXIIa catalysis activity were jointly correlated with the cores, specific surface areas, and curvatures of the NPs. CONCLUSIONS The heterogeneity of the NPs in their interactions with the proteins would be very promising in guiding the bioapplication of the nanotechnology. In this study, four kinds of NPs with different cores (Au, Ag and SiO2) and particle sizes (5 nm and 20 nm), apparently exhibited distinct characteristics in their interactions with plasma FXII and its activated form, -FXIIa. GNP-5 could adsorb FXII in the standing-up mode, and tightly bind with the heavy chain without inducing its conformational changes or cleavage. The conformation of FXII adsorbed on the other three NPs in the lying-down mode was obviously changed, resulting in the zymogen activation and 23


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

further cleavage in specific surface area dependent manners. The cleavage characterization showed that SNPs and SiNP-20 had different cutting sites. Moreover, in contrast to the respective inhibition effects of GNP-5 and SNP-5 on -FXIIa in noncompetitive and competitive modes, NPs with large particle sizes (i.e. SNP-20 and SiNP-20) would induce the favorable conformation change of -FXIIa for the catalysis of the substrate. Altogether, the findings herein, on one hand, firstly revealed the potential interactions of different NPs with plasma proteases, on the other hand, provided the important hints for tuning the key factors of the NPs in regulating the conformational and functional changes of target biological molecules, which might show a promise in nanodrug design.

24


Page 24 of 51

Page 25 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

MATERIALS AND METHODS Materials. GNP-5, SNP-5 and SNP-20 dispersed in sodium citrate aqueous solution (1 mg/mL), and SiNP-20 dispersed in silanol (Si–OH) aqueous solution (1 mg/mL) were bought from Nanocomposix Company (USA). No extra surfactant corona was introduced in any of the tested NPs, and their quality guarantee periods were 1 year when stored at 4 C in darkness. They remained monodisperse during the whole study, and no obvious aggregation was observed. The working solutions were freshly prepared by diluting the stock solution with distilled water. GNP-20 dispersed in sodium citrate solution (1 mg/mL) was obtained from the same vendor described above, and used for protein adsorption study. Both purified human FXII and -FXIIa were obtained from Enzyme Research Laboratories (USA). They were reconstituted in Milli-Q ultrapure water, and the aliquoted samples were stored at -80 ℃ till use. All the other chemicals or reagents with analytical grade purity or higher, were purchased from Sigma-Aldrich, unless otherwise specified. Characterization of the NPs and NP-FXII Bioconjugates. All NPs were characterized

to

obtain

their

physicochemical

properties

of

morphology,

hydrodynamic size and zeta potential. Briefly, 3 μL of NP dispersion was dropped onto a 230-mesh carbon film-coated copper grid, and dried overnight. The as-prepared samples were submitted to TEM observation at the accelerating voltage of 200 kV (JEOL, 2100F, Japan). The surface zeta potential and hydrodynamic sizes were evaluated by a Malvern Zetasizer Nano ZS (Marlvern, UK) at room temperature (i.e. 25 ℃), and the measurements were repeated for at least three times. 25


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

As for the characterization of NP-FXII bioconjugates, 2.5 μL of 50 μg/mL NP solutions were added to 25 μL of 0.4 μM FXII solution, and after 120 min incubation at 25 ℃, the NP-FXII bioconjugates were washed with phosphate buffer solution (PBS) for 3 times with centrifugation, dispersed in 1 mL of PBS, and finally submitted to DLS measurement at room temperature using a Malvern Zetasizer Nano ZS (Marlvern, UK). Each sample was measured for 3 times or more, and at least 1.5 × 1011 particles were tested for each measurement. The hydrodynamic radius of FXII was measured at 25 ℃ in the similar way described above by using 10 nM protein samples, and the mean effective diameter was determined by considering the spherical shape of the protein in the monodisperse system. The morphology of FXII protein was characterized by AFM (Bruker FastScan Bio, Germany), using the protocol with minor modification.66 Briefly, 5 μL of 1 μg/mL FXII was dropped onto the freshly cleaved mica pre-coated by 10 mM MgCl2 overnight, and incubated for 5 min. The protein-loaded mica was submitted to 3-time PBS wash, 1-time Milli-Q water (Millipore, USA) wash, and nitrogen drying in sequence. AFM imaging was carried out in tapping mode with silicon cantilevers (spring constant of 42 N/m, Nanosensor, Switzerland), and the subsequent analysis was performed using the built-in software (Nanoscope Analysis, v 1.80). Estimating the Adsorption Density of FXII on the NPs. The adsorption of FXII on NP surface was an assembling process, yielding NP-FXII bioconjugates. To calculate the maximum number of FXII adsorbed on the surface of each NP (Nmax), the following assumptions about the NP and protein were made. Firstly, the NP was 26


Page 26 of 51

Page 27 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

supposed to be modeled as perfect sphere, and its size should be equal. Secondly, the protein adsorbed on the NP was distributed evenly, and no steric hindrance effect existed between the adjacent protein molecules. Thirdly, the protein was in a spherical shape to simplify the calculation. The theoretic number of the protein adsorbed on the NPs was estimated according to the following equation (1).33 Nmax =

0.65(R3NP - FXII - R3NP) R3FXII

(1)

Where, RNP-FXII is the radius of NP-FXII bioconjugate, calculated by half of its hydrodynamic size. RNP and RFXII are those of the NPs and FXII, respectively. In this expression, FXII molecule was assumed to exist in the hard corona, closely packing around the NPs. The filling factor of 0.65 was also considered, and used to adjust the volume ratios, regarding the possible empty space between the adjacent protein molecules on the NPs.67 The adsorption density of FXII on the NPs was calculated by equation (2): DFXII =

Nmax 4πR2NP

(2)

The Deflection Angle between FXII and NPs. The average deflection angle was related with the radius of the NPs, and used to define the spatial arrangement of FXII. To calculate the average deflection angle between FXII on NP surface, FXII was modeled as a rod-like structure, and its footprint (i.e. the projection of 3D structure of FXII on NP surface) was circular. The related calculations were obtained by equation (3-5).38 K = R =

4πRNP2

(3)

Nmax K

(4)

π

27


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Deflection angle () =

Page 28 of 51

[ ]× 2R

180

RNP

π

(5)

Where, RNP represents the radius of the NPs. Nmax is the maximum number of FXII adsorbed on the NPs. K represents the surface area (nm2) occupied by each adsorbed FXII, and R is the radius of the circular footprint from FXII projection on NP surface. Evaluating the Adsorption Geometry of FXII on the NPs. The adsorption geometry of FXII on the NPs could be evaluated by the calculation of the protein semi-axis ratios, which were related with both longitudinal (i.e. vertical to the NP surface) and latitudinal (i.e. parallel to the NP surface) sizes of FXII in the NP-FXII bioconjugates. The former values were derived from half of the hydrodynamic diameter changes (ΔD) of FXII-NP complexes relative to the corresponding NPs. The latter data could be evaluated by the radii of the circular footprints from FXII projections on NP surfaces (R, equation 4). The semi-axis ratio of FXII was calculated by equation (6). Semi - axis ratio =

ΔD 4R

(6)

The longitudinal adsorption orientation of FXII (i.e. standing-up mode) was deduced when the semi-axes ratio was higher than 1, while the latitudinal adsorption orientation (i.e. lying-down mode) was concluded when the semi-axis ratio was lower than 1. NP-FXII Binding Affinity Assay. The binding affinity between the NPs and FXII was evaluated using a Monolith NT.115 (Nanotemper, Germany) based on MST, due to its high sensitivity and the low requirement of sample amount.68 Briefly, 4.3 μM FXII in MST optimized buffer (50 mM Tris-HCl containing 150 mM NaCl, 10 28


Page 29 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

mM MgCl2 and 0.05% Tween-20, pH 7.4) was submitted to the labeling process, which included the buffer replacement using the recommended Monolith NT Protein Labeling Kit (Nanotemper, Germany). After 30 min incubation at 25 C, the labeled protein was diluted 4 times with MST buffer, and used to titrate different concentrations of GNP-5 (0.0054-88.12 pM), SNP-5 (0.0076-62.5 pM), SNP-20 (0.0002-0.81 pM) or SiNP-20 (0.0029-23.87 pM), respectively, at the volume ratio of 1:1. The mixtures were incubated at 25 ℃ for 30 min, then loaded into premium capillaries (Nanotemper, Germany), and measured for fluorescence responses (Fnorm) using microscale thermophoresis with the illumination of 80% LED power and 20% IR-laser power. There were four stages of thermodiffusion, including fluorescence initial, fluorescence dropping, near steady-state, and diffusion back into original zone during the measurement. Based on MST curves of the NP-FXII bioconjugates, the normalized Fnorm was plotted against the NP concentration, and the binding affinity (Ka, nM) and dissociation constant (Kd, nM-1) were analyzed by MO affinity analysis software (v2.2.4, Nanotemper, Germany). The Gibbs free energy (ΔG, Jmol-1) was calculated by the following equation (7):69 ΔG = -RTlnKa = RTlnKd

(7)

HDX-MS Analysis for the Interaction Domains of FXII with the NPs. HDX-MS experiment was carried on an Acquity UPLC M-Class system with HDX-2 Automation (LEAP Technologies, USA) coupled with Synapt G2-Si HDMS (Waters, USA). The FXII powder was hydrated into Hepes buffer (Gibco, pH 7.4) and diluted to 0.4 μM. GNP-5, SNP-5, SNP-20 or SiNP-20 (20 μg/mL) were used for the 29


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 51

incubation with FXII solution for 30 min at 25 ℃. The bioconjugates were centrifuged to remove the excessive protein in the supernatant. The pellets were re-suspended by labeling buffer (100 mM sodium phosphate in D2O, pD 6.4) at an approximate 13-fold dilution for 10 s online exchange. The chilled quenching buffer (100 mM sodium phosphate, pH 2.3) was subsequently added to stop the exchange reaction at 0 ℃. To avoid excessive activation (i.e. the transformation of FXII to FXIIa) during incubation and quenching reaction, the specific areas of NPs were below 5 m2/g, and all vessels were prohibited from glass. After centrifugation, the samples passed through the desalination column (Acquity UPLC BEH, Waters Corporation, USA) and immobilized pepsin column (Enzymate Pepsin column, Waters Corporation, USA) for 4 min in 0.1% formic acid at a flow rate of 100 μL/min. Mass spectra were acquired in MSE mode over the mass range of 100 to 2000. HDX-MS data were processed by DynamX software, and the relative deuterium exchange rate was calculated by following equation (8):70

D% =

m – mcontrol m10 – mcontrol

 100%

(8)

Where, D% is the relative rate of deuterium exchange. The parameters, m and m10, are the average m/z values of the centroid mass peaks from the determined peptides of deuterated FXII with or without the incubation of the NPs, respectively. mcontrol is that of the corresponding peptide of FXII without deuteration. The D% less than 90% shows the insufficient hydrogen/deuterium exchange of the peptides due to their binding with the NPs. The D% higher than 110% suggests excessive 30


Page 31 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

hydrogen/deuterium exchange of the peptides because of NP-induced conformational changes of the protein. The D% in the range of 90-110% indicates that little interactions occur between the NPs and the tested peptides. Spectroscopic Analysis of Conformational Changes in FXII. 0.4 μM FXII solution was incubated with different specific surface areas of NPs at 25 ℃ for 0.5 h, and the fluorescence in the range of 290-450 nm was monitored on a FluoroMax fluorescence spectrophotometer (Horiba Scientific, USA) with the excitation wavelength of 278 nm using a 0.5 cm rectangular quartz cell (Starna, UK). The fluorescence intensities of FXII treated with a series of concentrations of GNP-5 were corrected for IFE,71 and plotted using Stern-Volmer model to give a linear curve of the ratio of F0/F against the concentration of the quencher.72 The following equation (9) was used to analyze the data from GNP-5 treatments. F0 F

= 1 + kqτ0[GNP - 5] = 1 + Ksv[GNP - 5]

(9)

Where, F0 and F are the maximum fluorescence intensities in the absence and presence of GNP-5, respectively. The parameter of kq is the quenching constant. τ0 is the life time of FXII chromophore in the absence of the quencher. Ksv is the Stern-Volmer quenching constant, and [GNP-5] is the molar concentration of GNP-5. As for the quantitative analysis of the fluorescence changes induced by the tested NPs, the emission intensities at 350 nm were recorded, and relative fluorescence values of F/F0 were evaluated against the specific areas of the NPs. The CD spectra of FXII (0.4 μM in PBS) in the absence or presence of the NPs at different specific areas (10 and 60 m2/g for GNP-5, SNP-5 and SiNP-20, 10 and 30 31


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

m2/g for SNP-20, respectively) were analyzed at 25 C on a Jasco J-815 spectropolarimeter (Japan) with a 0.5 cm rectangular quartz cell and 10 L/min nitrogen flux. After baseline correction, the spectra ranging from 250 to 200 nm were scanned at a speed of 100 nm/min with the interval of 1 nm, and each spectrum was the average of 3 measurements. The raw data in mdeg unit were transferred to ellipticity unit for further secondary content calculation with an online algorithm K2D2 package.31 Western Blot for FXII Cleavage. 25 μL of 0.4 μM FXII was incubated with different specific surface areas of NPs (3, 10, 20, 30, 40 and 60 m2/g GNP-5, SNP-5 and SiNP-20; 3, 10, 20 and 30 m2/g SNP-20) at 37 ℃ for 2 h. The negative and positive controls were prepared by the addition of PBS and 1 mg/mL kaolin, respectively. The as-prepared samples were subsequently submitted to Western blot, which briefly included protein separation in gradient gel (4%-20%, BioRad, Mini-PROTEAN, USA), the transfer to nitrocellulose membrane (Pall, USA), the immunoblotting using the primary antibody of anti-FXII (Cedarlane Laboratories, 1:2500, USA) and the secondary antibody of peroxidase-conjugated rabbit anti-goat (ZSGB-BIO, 1:3500, China), and the chemiluminescence photographic detection using ECL reagents (Pierce, USA). The pixel densities of the target protein bands in X-ray film was quantitatively calculated by ImageJ software (NIH, USA) after background subtraction. The enhanced chemiluminescence detection (Pierce, Thermo Fisher Scientific, USA) was also performed for Western blot of the as-prepared protein samples to analyze the trace levels of fragments from FXII cleavage due to NP 32


Page 32 of 51

Page 33 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

treatments. Ex vivo Experiment for FXII Activation. The whole human blood was collected from healthy donors, and 4% citrate sodium was used as the anticoagulant at the volume ratio of 1:9. The plasma was immediately isolated by 15 min centrifugation at 3,000 g, 4 ℃.73 The tested NPs at different specific surface areas (3, 10, 20, 30, 40 and 60 m2/g for GNP-5, SNP-5 and SiNP-20; 3, 10, 20 and 30 m2/g for SNP-20) were incubated with plasma samples at 37 ℃ for 2 h. The negative and positive controls were prepared by using PBS and 1 mg/mL kaolin, respectively. The above samples were subsequently submitted to Western blot for the evaluation of FXII cleavage. Enzymatic Activity Assay. The activity of the activated FXII (i.e. FXIIa) was measured in 25 μL of DPBS system, containing 0.4 μM FXII and 2.5 μL of the NPs at different specific surface areas (3, 10, 20, 30, 40 and 60 m2/g GNP-5, SNP-5 and SiNP-20; 3, 10, 20 and 30 m2/g SNP-20). After 2 h incubation at 37 ℃, 1 L of 8 mM chromogenic

substrate,

Pefachrome6017

(H-D-CHA-Gly-Arg-pNA2AcOH,

Pentapharm, Switzerland), was added, and the absorbance at 405 nm was immediately measured for the released chromophore (p-nitroaniline, pNA) using microplate reader (Thermo Scientific, VARIOSKAN FLASH ) for 45 min. The final enzymatic activities in different treatments were presented as the absorbance fold changes relative to that of the negative control. The “Pull-down” Assay. The tested NPs (0, 10, 20, 30, 40 and 60 m2/g GNP-5, SNP-5 and SiNP-20; 0, 10, 20 and 30 m2/g SNP-20) were incubated with 0.4 μM 33


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 51

FXII at 37 ℃ for 2 h, and the samples were subsequently centrifuged at 12,500 g for 15 min (4 ℃). 10 μL of supernatant was submitted to Western blot for FXII as described above. The pellets were washed with PBS for 3 times, and processed with the sampling buffer by 10 min heating at 95 ℃ for the denature of the protein bound on the NPs. The suspensions were finally characterized for the levels of FXII and its active form using the immunoblotting assay, according to the protocol mentioned above. The Kinetic Analysis of -FXIIa Activity upon NP treatments. This assay was performed by using the purified protein of -FXIIa (Figure S11). Namely, 0.4 μM -FXIIa was incubated with the NPs at different specific areas (30, 40 and 60 m2/g GNP-5, SNP-5 and SiNP-20, and 10, 20 and 30 m2/g SNP-20, respectively) at 37 ℃ for 0.5 h. The control was prepared by 0.4 μM -FXIIa without the addition of the NPs. The as-prepared samples were submitted to the enzymatic activity assay using Pefachrome6017 as the substrate. The concentration of substrate ranged from 25 μM to 400 μM, and the absorbance at 405 nm was instantly recorded for 30 min at the interval of 30 s. The initial velocity of the enzyme reaction (V0), represented by the slope of the kinetic curve of the enzyme reaction, was used to fit with Michaelis-Menten kinetic equation (10) for the calculation of Vmax and Km: V0 =

Vmax[S] Km + [S]

(10)

Where, Vmax is the maximum velocity of the enzyme reaction, [S] is the substrate concentration, and Km is the Michaelis-Menten constant. To further investigate the effect of the NPs on the catalytic activity of -FXIIa, 34


Page 35 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

the catalytic efficiency (Ceff) of enzyme in the absence and presence of the NPs at different specific surface areas was calculated following the equation (11).74 Ceff =

kcat Km

(11)

Where, kcat is the catalytic constant derived from the following equation (12). kcat =

Vmax

[ - FXIIa]

(12)

Where, [-FXIIa] is the concentration of -FXIIa. All kinetic parameters were processed using OriginPro8.5. Statistical Analysis. All assays were independently performed for at least three times, and the result was represented as mean ± standard deviation. Student’s t-test was used for the statistical analysis, and the significant difference was denoted by p value less than 0.05 (*) or 0.01 (**). ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Figure S1. Characterization of the NPs. Figure S2. The hydrodynamic diameter of FXII molecule. Figure S3. The tapping mode of AFM image for FXII molecule. Table S1. The adsorption geometry of FXII on the NPs based on the protein semi-axis ratio. Figure S4. The adsorption geometry of FXII on GNP-20. Figure S5. Typical MST signal profiles for the binding of FXII with the NPs. Table S2. The relative deuteration for the identified peptides of FXII upon NP incubation. Figure S6. The Stern-Volmer plot for the fluorescence of FXII incubated with GNP-5 after the 35


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 51

correction of IFE. Table S3. The Stern-Volmer quenching constants of FXII with GNP-5 treatment. Figure S7. FXII activation induced by SNP-5, SNP-20 and SiNP-20. Figure S8. FXII activation in human plasma induced by NP treatments. Figure S9. The schematic illustration for FXIIa catalysis of Pefachrome6017 substrate. Figure S10. Diverse cleavage sites of FXII in different NP treatments. Figure S11. Western blots for the purified human FXII and -FXIIa. Table S4. The influences of the NPs on the enzymatic kinetic parameters of -FXIIa. Figure S12. The schematic diagram for the interaction modes of the NPs with -FXIIa in its catalysis reaction. The authors declare no competing financial interest. AUTHOR INFORMATION Corresponding Author *Email: [email protected] ACKNOWLEDGEMENT We thank Dr. Xi Chen in Waters Corporation, Asia Pacific Headquarter for her support in HDX-MS experiment and data analysis. This study was financially supported by National Natural Science Foundation of China (21477153, 21621064, 21522706), Major International (Regional) Joint Project (21461142001),

and

the

Chinese

Academy

of

Science

(14040302,

QYZDJ-SSW-DQC017). REFERENCES 1. Chiarelli, P. A.; Revia, R. A.; Stephen, Z. R.; Wang, K.; Jeon, M.; Nelson, V.; Kievit, F. M.; Sham, J.; Ellenbogen, R. G.; Kiem, H.-P.; Zhang, M. Nanoparticle Biokinetics in Mice and Nonhuman Primates. ACS Nano 2017, 11, 9514-9524. 2. Betzer, O.; Perets, N.; Angel, A.; Motiei, M.; Sadan, T.; Yadid, G.; Offen, D.; 36


Page 37 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Popovtzer, R. In Vivo Neuroimaging of Exosomes Using Gold Nanoparticles. ACS Nano 2017, 11, 10883-10893. 3. Chen, W.-H.; Luo, G.-F.; Lei, Q.; Hong, S.; Qiu, W.-X.; Liu, L.-H.; Cheng, S.-X.; Zhang, X.-Z. Overcoming the Heat Endurance of Tumor Cells by Interfering with the Anaerobic Glycolysis Metabolism for Improved Photothermal Therapy. ACS Nano 2017, 11, 1419-1431. 4. Zhao, X.; Yang, C.-X.; Chen, L.-G.; Yan, X.-P. Dual-Stimuli Responsive and Reversibly Activatable Theranostic Nanoprobe for Precision Tumor-Targeting and Fluorescence-Guided Photothermal Therapy. Nat. Commun. 2017, 8, 14998. 5. Handy, R. D.; Shaw, B. J. Toxic Effects of Nanoparticles and Nanomaterials: Implications for Public Health, Risk Assessment and the Public Perception of Nanotechnology. Health Risk Soc. 2007, 9, 125-144. 6. Gwinn, M. R.; Vallyathan, V. Nanoparticles: Health Effects—Pros and Cons. Environ. Health Perspect. 2006, 114, 1818-1825. 7. Cedervall, T.; Lynch, I.; Lindman, S.; Berggård, T.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Understanding the Nanoparticle–Protein Corona Using Methods to Quantify Exchange Rates and Affinities of Proteins for Nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 2050-2055. 8. Lynch, I.; Cedervall, T.; Lundqvist, M.; Cabaleiro-Lago, C.; Linse, S.; Dawson, K. A. The Nanoparticle–Protein Complex as a Biological Entity; a Complex Fluids and Surface Science Challenge for the 21st Century. Adv. Colloid Interface Sci. 2007, 134, 167-174. 9. Zhang, B.; Xing, Y.; Li, Z.; Zhou, H.; Mu, Q.; Yan, B. Functionalized Carbon Nanotubes Specifically Bind to -Chymotrypsin’s Catalytic Site and Regulate Its Enzymatic Function. Nano Lett. 2009, 9, 2280-2284. 10. Wang, J.; Jensen, U. B.; Jensen, G. V.; Shipovskov, S.; Balakrishnan, V. S.; Otzen, D.; Pedersen, J. S.; Besenbacher, F.; Sutherland, D. S. Soft Interactions at Nanoparticles Alter Protein Function and Conformation in a Size Dependent Manner. Nano Lett. 2011, 11, 4985-4991. 11. Sanfins, E.; Dairou, J.; Hussain, S.; Busi, F.; Chaffotte, A. F.; Rodrigues-Lima, F.; Dupret, J.-M. Carbon Black Nanoparticles Impair Acetylation of Aromatic Amine Carcinogens through Inactivation of Arylamine N-Acetyltransferase Enzymes. ACS Nano 2011, 5, 4504-4511. 12. Decuzzi, P. Facilitating the Clinical Integration of Nanomedicines: The Roles of Theoretical and Computational Scientists. ACS Nano 2016, 10, 8133-8138. 13. Casals, E.; Pfaller, T.; Duschl, A.; Oostingh, G. J.; Puntes, V. Time Evolution of the Nanoparticle Protein Corona. ACS Nano 2010, 4, 3623-3632. 14. Lundqvist, M.; Stigler, J.; Cedervall, T.; Berggård, T.; Flanagan, M. B.; Lynch, I.; Elia, G.; Dawson, K. The Evolution of the Protein Corona around Nanoparticles: A Test Study. ACS Nano 2011, 5, 7503-7509. 15. Wang, Z.; Wang, C.; Liu, S.; He, W.; Wang, L.; Gan, J.; Huang, Z.; Wang, Z.; Wei, H.; Zhang, J.; Dong, L. Specifically Formed Corona on Silica Nanoparticles Enhances Transforming Growth Factor 1 Activity in Triggering Lung Fibrosis. ACS Nano 2017, 11, 1659-1672. 37


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

16. Huang, H.; Lai, W.; Cui, M.; Liang, L.; Lin, Y.; Fang, Q.; Liu, Y.; Xie, L. An Evaluation of Blood Compatibility of Silver Nanoparticles. Sci. Rep. 2016, 6, 25518. 17. Lacerda, S. H. D. P.; Park, J. J.; Meuse, C.; Pristinski, D.; Becker, M. L.; Karim, A.; Douglas, J. F. Interaction of Gold Nanoparticles with Common Human Blood Proteins. ACS Nano 2009, 4, 365-379. 18. Long, Y.-M.; Zhao, X.-C.; Clermont, A. C.; Zhou, Q.-F.; Liu, Q.; Feener, E. P.; Yan, B.; Jiang, G.-B. Negatively Charged Silver Nanoparticles Cause Retinal Vascular Permeability by Activating Plasma Contact System and Disrupting Adherens Junction. Nanotoxicology 2016, 10, 501-511. 19. Simberg, D.; Zhang, W.-M.; Merkulov, S.; McCrae, K.; Park, J.-H.; Sailor, M. J.; Ruoslahti, E. Contact Activation of Kallikrein–Kinin System by Superparamagnetic Iron Oxide Nanoparticles in Vitro and in Vivo. J. Controlled Release 2009, 140, 301-305. 20. Ekstrand-Hammarström, B.; Hong, J.; Davoodpour, P.; Sandholm, K.; Ekdahl, K. N.; Bucht, A.; Nilsson, B. TiO2 Nanoparticles Tested in a Novel Screening Whole Human Blood Model of Toxicity Trigger Adverse Activation of the Kallikrein System at Low Concentrations. Biomaterials 2015, 51, 58-68. 21. Ekdahl, K. N.; Davoodpour, P.; Ekstrand-Hammarström, B.; Fromell, K.; Hamad, O. A.; Hong, J.; Bucht, A.; Mohlin, C.; Seisenbaeva, G. A.; Kessler, V. G. Contact (Kallikrein/Kinin) System Activation in Whole Human Blood Induced by Low Concentrations of -Fe2O3 Nanoparticles. Nanomedicine (N. Y., NY, U. S.) 2018, 14, 735-744. 22. Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K. A. Nanoparticle Size and Surface Properties Determine the Protein Corona with Possible Implications for Biological Impacts. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 14265-14270. 23. Chakraborty, S.; Joshi, P.; Shanker, V.; Ansari, Z.; Singh, S. P.; Chakrabarti, P. Contrasting Effect of Gold Nanoparticles and Nanorods with Different Surface Modifications on the Structure and Activity of Bovine Serum Albumin. Langmuir 2011, 27, 7722-7731. 24. Meder, F.; Daberkow, T.; Treccani, L.; Wilhelm, M.; Schowalter, M.; Rosenauer, A.; Mädler, L.; Rezwan, K. Protein Adsorption on Colloidal Alumina Particles Functionalized with Amino, Carboxyl, Sulfonate and Phosphate Groups. Acta Biomater. 2012, 8, 1221-1229. 25. Cho, E. C.; Zhang, Q.; Xia, Y. The Effect of Sedimentation and Diffusion on Cellular Uptake of Gold Nanoparticles. Nat. Nanotechnol. 2011, 6, 385-391. 26. Vilanova, O.; Mittag, J. J.; Kelly, P. M.; Milani, S.; Dawson, K. A.; Rädler, J. O.; Franzese, G. Understanding the Kinetics of Protein–Nanoparticle Corona Formation. ACS Nano 2016, 10, 10842-10850. 27. Setyawati, M. I.; Tay, C. Y.; Bay, B. H.; Leong, D. T. Gold Nanoparticles Induced Endothelial Leakiness Depends on Particle Size and Endothelial Cell Origin. ACS Nano 2017, 11, 5020-5030. 28. Tang, S.; Peng, C.; Xu, J.; Du, B.; Wang, Q.; Vinluan, R. D.; Yu, M.; Kim, M. J.; Zheng, J. Tailoring Renal Clearance and Tumor Targeting of Ultrasmall Metal 38


Page 38 of 51

Page 39 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Nanoparticles with Particle Density. Angew. Chem. 2016, 128, 16273-16277. 29. Bryant, J.; Shariat-Madar, Z. Human Plasma Kallikrein-Kinin System: Physiological and Biochemical Parameters. Cardiovasc. Hematol. Agents Med. Chem. 2009, 7, 234-250. 30. Tay, C. Y.; Setyawati, M. I.; Leong, D. T. Nanoparticle Density: A Critical Biophysical Regulator of Endothelial Permeability. ACS Nano 2017, 11, 2764-2772. 31. Huang, R. X.; Carney, R. R.; Ikuma, K.; Stellacci, F.; Lau, B. L. T. Effects of Surface Compositional and Structural Heterogeneity on Nanoparticle-Protein Interactions: Different Protein Configurations. ACS Nano 2014, 8, 5402-5412. 32. Sanfins, E.; Augustsson, C.; Dahlbäck, B.; Linse, S.; Cedervall, T. Size-Dependent Effects of Nanoparticles on Enzymes in the Blood Coagulation Cascade. Nano Lett. 2014, 14, 4736-4744. 33. Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C.; Mikulec, F. V.; Bawendi, M. G. Self-Assembly of CdSe−ZnS Quantum Dot Bioconjugates Using an Engineered Recombinant Protein. J. Am. Chem. Soc. 2000, 122, 12142-12150. 34. Mahmoudi, M.; Lynch, I.; Ejtehadi, M. R.; Monopoli, M. P.; Bombelli, F. B.; Laurent, S. Protein−Nanoparticle Interactions: Opportunities and Challenges. Chem. Rev. (Washington, DC, U. S.) 2011, 111, 5610-5637. 35. Li, N.; Zeng, S.; He, L.; Zhong, W. Probing Nanoparticle−Protein Interaction by Capillary Electrophoresis. Anal. Chem. (Washington, DC, U. S.) 2010, 82, 7460-7466. 36. Roach, P.; Farrar, D.; Perry, C. C. Surface Tailoring for Controlled Protein Adsorption: Effect of Topography at the Nanometer Scale and Chemistry. J. Am. Chem. Soc. 2006, 128, 3939-3945. 37. Treuel, L.; Brandholt, S.; Maffre, P.; Wiegele, S.; Shang, L.; Nienhaus, G. U. Impact of Protein Modification on the Protein Corona on Nanoparticles and Nanoparticle–Cell Interactions. ACS Nano 2014, 8, 503-513. 38. Hill, H. D.; Millstone, J. E.; Banholzer, M. J.; Mirkin, C. A. The Role Radius of Curvature Plays in Thiolated Oligonucleotide Loading on Gold Nanoparticles. ACS Nano 2009, 3, 418-424. 39. Lara, S.; Alnasser, F.; Polo, E.; Garry, D.; Lo Giudice, M. C.; Hristov, D. R.; Rocks, L.; Salvati, A.; Yan, Y.; Dawson, K. A. Identification of Receptor Binding to the Biomolecular Corona of Nanoparticles. ACS Nano 2017, 11, 1884-1893. 40. Wienken, C. J.; Baaske, P.; Rothbauer, U.; Braun, D.; Duhr, S. Protein-Binding Assays in Biological Liquids Using Microscale Thermophoresis. Nat. Commun. 2010, 1, 100. 41. Buijs, J.; Ramström, M.; Danfelter, M.; Larsericsdotter, H.; Håkansson, P.; Oscarsson, S. Localized Changes in the Structural Stability of Myoglobin Upon Adsorption onto Silica Particles, as Studied with Hydrogen/Deuterium Exchange Mass Spectrometry. J. Colloid Interface Sci. 2003, 263, 441-448. 42. Roach, P.; Farrar, D.; Perry, C. C. Interpretation of Protein Adsorption: Surface-Induced Conformational Changes. J. Am. Chem. Soc. 2005, 127, 8168-8173. 43. Lundqvist, M.; Sethson, I.; Jonsson, B.-H. Protein Adsorption onto Silica Nanoparticles: Conformational Changes Depend on the Particles' Curvature and the 39


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Protein Stability. Langmuir 2004, 20, 10639-10647. 44. Subramanyam, R.; Gollapudi, A.; Bonigala, P.; Chinnaboina, M.; Amooru, D. G. Betulinic Acid Binding to Human Serum Albumin: A Study of Protein Conformation and Binding Affinity. J. Photochem. Photobiol., B 2009, 94, 8-12. 45. Zhao, X.; Lu, D.; Liu, S. Q.; Li, Y.; Feng, R.; Hao, F.; Qu, G.; Zhou, Q.; Jiang, G. Hematological Effects of Gold Nanorods on Erythrocytes: Hemolysis and Hemoglobin Conformational and Functional Changes. Adv. Sci. (Weinheim, Ger.) 2017, 4, 1700296. 46. Sandros, M. G.; Gao, D.; Gokdemir, C.; Benson, D. E. General, High-Affinity Approach for the Synthesis of Fluorophore Appended Protein Nanoparticle Assemblies. Chem. Commun. (Cambridge, U. K.) 2005, 22, 2832-2834. 47. Hao, F.; Jing, M.; Zhao, X.; Liu, R. Spectroscopy, Calorimetry and Molecular Simulation Studies on the Interaction of Catalase with Copper Ion. J. Photochem. Photobiol., B 2015, 143, 100-106. 48. Liu, Q. S.; Sun, Y.; Qu, G.; Long, Y.; Zhao, X.; Zhang, A.; Zhou, Q.; Hu, L.; Jiang, G. Structure-Dependent Hematological Effects of Per-and Polyfluoroalkyl Substances on Activation of Plasma Kallikrein–Kinin System Cascade. Environ. Sci. Technol. 2017, 51, 10173-10183. 49. Zhao, X.; Hao, F.; Lu, D.; Liu, W.; Zhou, Q.; Jiang, G. Influence of the Surface Functional Group Density on the Carbon-Nanotube-Induced -Chymotrypsin Structure and Activity Alterations. ACS Appl. Mater. Interfaces 2015, 7, 18880-18890. 50. Papadopoulou, A.; Green, R. J.; Frazier, R. A. Interaction of Flavonoids with Bovine Serum Albumin: A Fluorescence Quenching Study. J. Agric. Food Chem. 2005, 53, 158-163. 51. Mariam, J.; Dongre, P.; Kothari, D. Study of Interaction of Silver Nanoparticles with Bovine Serum Albumin Using Fluorescence Spectroscopy. J. Fluoresc. 2011, 21, 2193-2199. 52. Bardhan, R.; Grady, N. K.; Cole, J. R.; Joshi, A.; Halas, N. J. Fluorescence Enhancement by Au Nanostructures: Nanoshells and Nanorods. ACS Nano 2009, 3, 744-752. 53. Pan, B.; Cui, D.; Xu, P.; Li, Q.; Huang, T.; He, R.; Gao, F. Study on Interaction between Gold Nanorod and Bovine Serum Albumin. Colloids Surf., A 2007, 295, 217-222. 54. Samuel, M.; Samuel, E.; Villanueva, G. B. The Low pH Stability of Human Coagulation Factor XII (Hageman Factor) Is Due to Reversible Conformational Transitions. Thromb. Res. 1994, 75, 259-268. 55. Zhao, X.; Lu, D.; Hao, F.; Liu, R. Exploring the Diameter and Surface Dependent Conformational Changes in Carbon Nanotube-Protein Corona and the Related Cytotoxicity. J. Hazard. Mater. 2015, 292, 98-107. 56. Konings, J.; Govers-Riemslag, J. W.; Philippou, H.; Mutch, N. J.; Borissoff, J. I.; Allan, P.; Mohan, S.; Tans, G.; Ten Cate, H.; Ariens, R. A. Factor XIIa Regulates the Structure of the Fibrin Clot Independently of Thrombin Generation through Direct Interaction with Fibrin. Blood 2011, 118, 3942-3951. 40


Page 40 of 51

Page 41 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

57. Stavrou, E.; Schmaier, A. H. Factor XII: What Does It Contribute to Our Understanding of the Physiology and Pathophysiology of Hemostasis & Thrombosis. Thromb. Res. 2010, 125, 210-215. 58. Dementiev, A.; Silva, A.; Yee, C.; Li, Z.; Flavin, M. T.; Sham, H.; Partridge, J. R. Structures of Human Plasma Beta-Factor XIIa Cocrystallized with Potent Inhibitors. Blood Adv. 2018, 2, 549-558. 59. Colman, R. W.; Schmaier, A. H. Contact System: A Vascular Biology Modulator with Anticoagulant, Profibrinolytic, Antiadhesive, and Proinflammatory Attributes. Blood 1997, 90, 3819-3843. 60. Dunn, J. T.; Silverberg, M.; Kaplan, A. P. The Cleavage and Formation of Activated Human Hageman Factor by Autodigestion and by Kallikrein. J. Biol. Chem. 1982, 257, 1779-1784. 61. Hovinga, J.; Schaller, J.; Stricker, H.; Wuillemin, W.; Furlan, M.; Lammle, B. Coagulation Factor XII Locarno: The Functional Defect Is Caused by the Amino Acid Substitution Arg 353Pro Leading to Loss of a Kallikrein Cleavage Site. Blood 1994, 84, 1173-1181. 62. Nelson, D. L.; Cox, M. M. Enzymes. In Lehninger Principles of Biochemistry; Nelson, D. L., Lehninger, A. L., Cox, M. M., Eds.; WH Freeman and Company: New York, 2005; pp 205-212. 63. Kushida, T.; Saha, K.; Subramani, C.; Nandwana, V.; Rotello, V. M. Effect of Nano-Scale Curvature on the Intrinsic Blood Coagulation System. Nanoscale 2014, 6, 14484-14487. 64. Chen, X.; Wang, J.; Paszti, Z.; Wang, F.; Schrauben, J. N.; Tarabara, V. V.; Schmaier, A. H.; Chen, Z. Ordered Adsorption of Coagulation Factor XII on Negatively Charged Polymer Surfaces Probed by Sum Frequency Generation Vibrational Spectroscopy. Anal. Bioanal. Chem. 2007, 388, 65-72. 65. Griffin, J. H. Role of Surface in Surface-Dependent Activation of Hageman Factor (Blood Coagulation Factor XII). Proc. Natl. Acad. Sci. U. S. A. 1978, 75, 1998-2002. 66. Basu, D.; Khare, G.; Singh, S.; Tyagi, A.; Khosla, S.; Mande, S. C. A Novel Nucleoid-Associated Protein of Mycobacterium Tuberculosis Is a Sequence Homolog of Groel. Nucleic Acids Res. 2009, 37, 4944-4954. 67. Cebula, D. J.; Ottewill, R. H.; Ralston, J.; Pusey, P. N. Investigations of Microemulsions by Light Scattering and Neutron Scattering. J. Chem. Soc., Faraday Trans. 1 1981, 77, 2585-2612. 68. Zhu, Q.; Li, T.; Ma, Y.; Wang, Z.; Huang, J.; Liu, R.; Gu, Y. Colorimetric Detection of Cholic Acid Based on an Aptamer Adsorbed Gold Nanoprobe. RSC Adv. 2017, 7, 19250-19256. 69. Hu, Y.-J.; Liu, Y.; Xiao, X.-H. Investigation of the Interaction between Berberine and Human Serum Albumin. Biomacromolecules 2009, 10, 517-521. 70. Smith, D. L.; Deng, Y.; Zhang, Z. Probing the Non-Covalent Structure of Proteins by Amide Hydrogen Exchange and Mass Spectrometry. J. Mass Spectrom. 1997, 32, 135-146. 71. Kubista, M.; Sjöback, R.; Eriksson, S.; Albinsson, B. Experimental Correction for 41


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the Inner-Filter Effect in Fluorescence Spectra. Analyst (Cambridge, United Kingdom) 1994, 119, 417-419. 72. Lacerda, S. H. D. P.; Park, J. J.; Meuse, C.; Pristinski, D.; Becker, M. L.; Karim, A.; Douglas, J. F. Interaction of Gold Nanoparticles with Common Human Blood Proteins. ACS Nano 2010, 4, 365-379. 73. Tenzer, S.; Docter, D.; Rosfa, S.; Wlodarski, A.; Kuharev, J.; Rekik, A.; Knauer, S. K.; Bantz, C.; Nawroth, T.; Bier, C.; Sirirattanapan, J.; Mann, W.; Treuel, L.; Zellner, R.; Maskos, M.; Schild, H.; Stauber, R. H. Nanoparticle Size Is a Critical Physicochemical Determinant of the Human Blood Plasma Corona: A Comprehensive Quantitative Proteomic Analysis. ACS Nano 2011, 5, 7155-7167. 74. Rubinow, S.; Lebowitz, J. L. Time-Dependent Michaelis-Menten Kinetics for an Enzyme-Substrate-Inhibitor System. J. Am. Chem. Soc. 1970, 92, 3888-3893.

42


Page 42 of 51

Page 43 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

FIGURE LEGENDS Figure 1. Characterization of FXII adsorption on the tested NPs based on DLS measurement and theoretical calculation. (A) The hydrodynamic diameters of bare NPs and NP-protein complexes (n = 3). The error bars represent the standard deviations. *p < 0.05; **p < 0.01. (B) The adsorption parameters of FXII on the tested NPs (n = 3). (C) The adsorption geometries and the related deflection angles of FXII molecules on the NPs. Left panel: The vertical (standing-up) mode of FXII adsorbed on GNP-5 with a small deflection angle. Middle and right panels: The horizontal (lying-down) mode of FXII adsorbed on SNP-5, SNP-20 or SiNP-20, respectively. Figure 2. MST profiles of the NPs upon FXII adsorption at 298 K. (A) The representative thermophoresis curves for a series of concentrations of GNP-5 with FXII adsorption. (B) Normalized fluorescence signals versus NP concentrations in thermophoretic diffusion assay (n = 3). The error bars represent the standard deviations. (C) The parameters for FXII adsorption on the NPs (n = 3). Figure 3. HDX-MS analysis for the binding sites of the NPs in FXII. (A) The workflow for HDX-MS analysis. (B) Representative isotopic MS data for different peptides of FXII with or without NP treatments. (C) The schematic graph for deuteration percentages of FXII at different domains upon NP treatments. The hierarchical clustering result depicted by the gray lines distinguished different binding modes of the tested NPs. Figure 4. Conformational alterations of FXII upon NP bioconjugation. (A) The representative fluorescence emission spectra of FXII with 20 m2/g NP treatments. 43


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(B) The specific surface area-related effects of the NPs on FXII fluorescence intensity at 350 nm (n = 3). The error bars represent the standard deviations. (C) The CD spectra of FXII upon NP treatments at different specific areas. The inserts represent the contents of the secondary structures of FXII obtained from the corresponding CD spectrum in different treatments. Figure 5. FXII activation induced by NP treatments. (A) Western blot results of FXII upon NP incubations at different specific areas. (B) The relative FXIIa activity in different NP treatments (n = 3). The error bars represent the standard deviations. (C) The schematic diagram of “Pull-down” assay. (D) Western blot analysis of the supernatant and pellet samples from the “Pull-down” assay. Figure 6. The enzymatic activity changes of -FXIIa upon NP treatments. (A) The fitting curves for the initial velocities of -FXIIa in different NP treatments. (n = 4). The error bars represent the standard deviations. (B) The enzymatic kinetic parameters of -FXIIa in different NP treatments.

44


Page 44 of 51

Page 45 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 1.

45


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2.

46


Page 46 of 51

Page 47 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 3.

47


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4.

48


Page 48 of 51

Page 49 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

Figure 5.

49


ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6.

50


Page 50 of 51

Page 51 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Nano

TOC

51


Exploring the Heterogeneity of Nanoparticles in Their Interactions with

Recommend Documents