Understanding the Chemical Nature of NanoparticleâProtein

Review Cite This: Bioconjugate Chem. 2019, 30, 1923−1937

pubs.acs.org/bc

Understanding the Chemical Nature of Nanoparticle−Protein Interactions Didar Baimanov,†,§,‡ Rong Cai,†,‡ and Chunying Chen*,†,§ †

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CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China § University of Chinese Academy of Sciences, Beijing 100049, China ABSTRACT: The formation of a protein corona has been considered a pitfall in the clinical translation of nanomedicines. Hence, interdisciplinary studies on corona characterization are critically essential. A deep understanding of the formation of hard and soft protein coronas upon in vivo administration of nanoparticles is vital. The protein corona gives the nanoplatform a new biological identity. Furthermore, the control of and mechanistic understanding of corona formation as it is regulated by the physicochemical properties of nanoparticles is crucial for developing safe nanomedicines. A growing number of analytical techniques have been developed in the past decade for examining NP−protein interactions, contributing to a better understanding of protein corona formation on the surface of nanoparticles. In this Review, we summarize the latest developments in the in vivo and in vitro study of dynamic protein corona formation. Insights derived from techniques used to visualize, quantify, and define protein coronas, as well as the methods for examining the kinetics and structural changes of coronal proteins, are discussed. The potential challenges and future perspectives in the study of protein corona formation and its effects on biological behavior and applications of therapeutic nanomaterials are also provided.

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INTRODUCTION In the past decade, it has been rigorously established that all particles administered into biological fluids are inevitably and immediately (>0.5 min) coated by biomolecules, such as enzymes, proteins, peptides, and/or amino acids, to form what is known as a “corona” on the particle surface. The protein corona was first described in 2007 by Dawson et al., who studied the proteins associated with nanoparticles (NPs) with a wide range of binding affinities in various biological fluids.1 The protein corona evolved to the “biomolecular corona” that surrounds NPs in 2012 by Monopoli et al.2 The formation process of the biomolecular corona is dynamic3 and depends on the unique properties of the NPs, such as size,4,5 surface chemistry, ligands,5 nature,6 shape,7 charge,8−10 surface curvature, and environmental impact.11,12 The nature and composition of the biomolecular corona around NPs may affect the biological fate,13,14 cellular uptake,15−18 immunological response,19,20 and biodistribution of NPs.9,16 After administration into the bloodstream, the corona composition of NPs dynamically shifts until an equilibrium reached. Dramatic changes take place on a “soft corona” due to the low affinity of proteins, while only small changes occur in a “hard corona”, where comparably stronger interactions bind the biomolecules onto a NP’s surface. The hard corona achieves equilibrium in seconds to 30 min,21 while a soft corona may require several hours.22 Nowadays, most analytical techniques focus on studying the hard corona; only a few examine the soft corona with real-time monitoring (Table 1). © 2019 American Chemical Society

Understanding the unique properties of the protein corona is essential to nanomedicine. This protein coating may slow the degradation of NPs, decrease their cytotoxicity,23−25 influence their in vivo half-life,26 change their uptake, stimulate their internalization by cells, and affect the inflammation signaling pathway.27,28 Due to the complexity of biomolecular coronas, it is essential that one uses appropriate and accurate methods to characterize the corona so as to understand the adsorption process, adsorption kinetics, and binding affinity, as well as quantify the adsorbed biomolecules. In addition, the nanoparticle−biology (nanobio) interface characterization is an important consideration. Human plasma consists of more than 3500 proteins, but not all of them will adsorb onto the surface of a given NP. The protein coronas formed around NPs possess different natures29,30 and usually endow the NPs with new biological effects31 and identity.2,32,33

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CHARACTERIZATION OF PROTEIN ADSORPTION ON NANOPARTICLES

Visualization and Thickness Quantification of Protein Corona Formation. Visualization methods, including atomic force microscopy (AFM) (Figure 1a),23,24,34−36 transmission electron microscopy (TEM) (Figure 1b),35,37−39 and scanning Received: May 15, 2019 Revised: June 16, 2019 Published: June 17, 2019 1923

DOI: 10.1021/acs.bioconjchem.9b00348 Bioconjugate Chem. 2019, 30, 1923−1937

Quartz Crystal Microbalance (QCM)

Inductive coupled plasma mass spectrometry (ICP-MS) Isothermal Titration Calorimetry (ITC) Surface Plasmon Resonance (SPR) Circular Dichroism (CD)

Quantification

Kinetic

In situ

1924

Fluorescence Correlation Spectroscopy (FCS) F NMR

19

Nuclear Magnetic Resonance (NMR)

Surface-enhanced Raman Scattering (SERS)

Raman Spectroscopy (RS)

Fourier Transform Infrared (FTIR)

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Mass Spectrometry (MS)

Identification

Conformation

Atomic Force Microscopy (AFM) Transmission Electron Microscopy (TEM) Scanning Electron Microscopy (SEM)

Techniques

Visualization

Characterizations

Limitations

Impossible to distinguish hard and/or soft corona formation Negative staining of protein is needed Difficulties in detection of proteins on the NP surface, staining with heavy metals is required Separation of proteins by molecular weight, quantification of Protein determination possible after isolation of NP−protein proteins in the different weight range coronas from the biological fluid Determination of protein corona content, gel and nongel based Separation of protein by trypsin digesting, complicated to methods analyze Highly sensitive to total mass change Impossible to distinguish the mass changes of specific molecules Quantification of the amount of metal (NPs) and serum (proteins Can quantify only inorganic NPs and calculate NP/protein and NPs) ratio only under known conditions Detection of proteins’ binding affinities Limitations on sample concentration (NP aggregation) Detection of NP and protein binding Comparably higher cost Real time secondary structure determination in the presence of Impossible to provide information about structural changes of unbound proteins individual amino acid residues Highly sensitive, possible to measure in liquid and solid states For measurements in the solid state, an isolation step is required Comparably simple, possible to detect aromatic chains, peptides Only metal NPs, Detection of Raman active molecules and sulfur bonds from protein Analysis of secondary structure changes due to protein adsorption Low sensitivity, impossible to detect carbon based NPs on NPs Dynamic interaction details of the structural changes of proteins Difficult to analyze NP−protein complexes and the chemical nature of molecules Real time detection of kinetic and thermodynamic changes Detection of fluorescent molecules Higher abundance compared to 13C and less signal overlap Complicated matrix of analyst compared to 1H

The real state of NPs, visualization of protein adsorption Visualization of protein adsorption onto NP surfaces Minimal preparation, can acquire detailed 3D images

Advantages

Table 1. Comparison of Analytical Techniques for Characterization of Protein Corona

43−45 75

52,71−73

71

70,71

63,67−69

59,61 4,23,60 23,24,60,64−66

49

50,51,63

4,5,46,47,49,56

4,39

23,24,34−36 35,37−39 17

ref.

Bioconjugate Chemistry Review


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Bioconjugate Chemistry

Figure 1. Visualization, quantification, and identification of NP−protein corona complexes by various analytical techniques. (a) Interaction between fibrinogen and graphene oxide captured by AFM. AFM images of NP−protein complexes were acquired after a 5 min incubation. (Adapted with permission from ref 23. Copyright 2015. American Chemical Society.) (b) TEM image of precoated ZnO NPs in a dry state. (Adapted with permission from ref 39. Copyright 2015. The Royal Society of Chemistry.) (c) SEM image and corresponding size distribution plot of HSA-precoated carboxylic acid-functionalized silica NPs after a 1 h incubation with human plasma. Scale bar: 500 nm. (Adapted with permission from ref 17. Copyright 2016. Elsevier.) (d) Representative TEM micrographs of immunogold labeling. Polymeric NPs with carboxyl surface groups were incubated with human serum and stained with a secondary, 12 nm colloidal gold−antibody. Scale bars: 200 nm. (Adapted with permission from ref 40. Copyright 2018. Nature Publishing Group.) (e) Adsorption of HSA, succinic anhydride, and ethylenediamine surface modified HSA onto dihydrolipoic acid-coated quantum dots. (Adapted with permission from ref 45. Copyright 2014. American Chemical Society.) (f) The protein adsorption capacity was calculated as the mass of protein adsorbed vs the mass ratio of protein to material by systematically increasing the ratio of protein to graphene oxide. (Adapted with permission from ref 23. Copyright 2015. American Chemical Society.) (g) Schematic illustration of the frequency changes (ΔF) in QCM-D measurements. The COOH-coated chip was activated by EDC/NHS and sequentially exposed to PenAu NPs (Step 1); transferrin (Tf) (Step 2); and HEK293 cell-derived liposomes containing three different levels of Tf receptor expression (knockdown, wide-type, and overexpressed; Step 3). (Adapted with permission from ref 63. Copyright 2017. American Chemical Society.) (h) SDS-PAGE of adsorbed protein species on pristine and precoated NPs after a 24 h incubation in serum-supplemented cell culture medium. (Adapted with permission from ref 39. Copyright 2015. The Royal Society of Chemistry.)

electron microscopy (SEM) (Figure 1c),17 have been used to detect protein coronas on NP surfaces. Chong et al.23 examined the adsorption of highly abundant blood proteins on graphene oxide (GO) and reduced GO (rGO) nanomaterials by AFM. Bovine serum albumin (BSA) formed complex aggregates during the incubation with GO. The protein distribution and particle morphology were visualized via AFM. The differences in the distribution of adsorbed proteins were attributed to the differences in particle structure and incubation time. The authors described noticeable conformational changes of the adsorbed proteins on GO. Similar to carbon-based nanomaterials, Ge et al.24 observed various binding morphologies of blood proteins onto carbon nanotubes (CNTs). The number of molecules of each protein on a single CNT was measured by AFM. Adsorption driven by π−π stacking of CNTs with proteins’ aromatic residues is a key feature of adsorption capacity. The thickness of the protein layer on the CNT surface was about 2.5 nm. AFM height analysis demonstrated a strong linear correlation between the hydrophobic amino acid content of the proteins and the amount of protein bound in the protein−CNT complex. Another novel technique to visualize and characterize the protein corona is immunogold labeling (Figure 1d). Gold NPs preadsorbed with human serum were stained with

secondary gold-coupled antibodies for visualization by TEM. Tonigold et al.40 showed that NPs preadsorbed with antibodies were not completely covered by the biomolecular corona. Most importantly, this technique can be used for specific protein detection due to the use of antibodies. Some components of the protein corona have natural fluorescence properties (e.g., tyrosine, tryptophan, and phenylalanine).41 Fluorescence-labeling techniques (FS) can be used to monitor protein−NP interactions. The adsorption of human serum albumin (HSA; 3.3 nm monolayer) on NPs and timeresolved HSA binding have been performed using FS,42 demonstrating that the FS technique can be used as a real time detection tool. Duan et al.43 demonstrated that fluorescence labeling can be used to determine binding affinity and conformational changes of proteins after protein−NP interactions. Notably, upon interaction with negatively charged NPs, the conformations of all the examined metal-proteins were dramatically changed.43 Fluorescence correlation spectroscopy (FCS; Figure 1e) is a powerful technique for detecting kinetic and thermodynamic changes in fluorescent or fluorescence-labeled molecules in solution without the need for removing unbound proteins.42,44,45 Treuel et al. used FCS to investigate the surface-modification of dihydrolipoic acidcoated quantum dots (QDs; ∼5 nm) by HSA adsorption. The 1925


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Figure 2. Illustration of hard corona formation on the surface of citrate-stabilized Au NPs of various sizes: (i) incomplete hard corona, (ii) a single dense protein corona layer, and (iii) multilayer corona formation. (Adapted with permission from ref 29. Copyright 2016. American Chemical Society.)

Quartz crystal microbalance (QCM; Figure 1g) is a technique based on the relationship between the viscosity and density of a solution to the adsorbed mass.50 Brewer et al. summarized the effects of surface modification of citrate-coated Au NPs on the binding mechanism of BSA in the following order: hydrophobic > COO− > NH3+ > OH > ethylene glycol.50 This technique is based on real-time frequency shifts when the NPs were located on an oscillating quartz surface; the association and dissociation constants can thus be determined. QCM has high sensitivity, where the frequency changes represent mass change (e.g., protein, water, and ion adsorption), while the presence of water and ions is ignored in the calculation of small protein adsorption.51 The adsorption of BSA, cytochrome C (CytC), and myoglobin (Mb) onto Au NPs was detected by quartz crystal microbalance measurements with dissipation (QCM-D). The highly adsorbed CytC protein covered nearly the entire Au NP surface in a monolayer protein corona.51 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Figure 1h) is based on the molecular weight of proteins. In the case of protein complexes, such as in a biological fluid, SDS-PAGE only provides the quantity of proteins in different weight ranges. Cedervall et al. separated apolipoprotein A-1 (ApoA-1) from 70 and 200 nm, plasmatreated copolymer NPs by SDS-PAGE. A strongly positive correlation between NP hydrophobicity and the adsorbed amount of ApoA-1 was observed.4 Several possible regimes of protein absorption onto NPs surface have been proposed (Figure 2): (i) incomplete corona (3 nm NPs), (ii) a near-single layer protein corona (10 nm NPs), and (iii) a multilayer corona (100 nm NPs).29 Soft Corona. Due to the rapidly growing number of nanobiomedical applications, an understanding of NP protein corona formation is crucial. The hard corona comprises strongly bound proteins, while the soft corona is dynamic and comprises loosely bound proteins whose binding reaches an equilibrium within the circulation. At this time, there are no established, specific criteria to distinguish soft and hard

succinic anhydride surface modified HSA presented decreasing binding affinity toward interaction with QDs, by forming a thick protein corona of ∼8.1 nm. In contrast, ethylenediamine modification elicited enhanced binding affinity by forming a protein corona with a thickness of 4.6 nm. The thickness of the native HSA-formed protein corona on QDs was around 3.3 nm. The authors concluded that the change in binding affinity of the protein for the NPs was due to additional carboxyl and amino group modified proteins.46 One advantage of this technique is that it offers an in situ method for protein corona size detection in complex systems; however, the requirement for fluorescence labeling may lead to deformation of the protein corona and its biological behavior. Walczyk et al. also studied the thickness of protein coronas under different in situ conditions by differential centrifugal sedimentation (DCS). DCS can be used to semiquantitatively assess size variations in NP−protein complexes in the presence of a complex protein mixture and with unbound or loosely bound proteins.46,47 Quantification of Protein Amounts in NP Protein Coronas. Protein quantification assays (e.g., bicinchoninic acid assay (BCA;48 Figure 1f; and Bradford assay) can be used to evaluate the amount of proteins adsorbed on NPs. The limitations of these approaches are their low sensitivity and the possible confounding effects of the NPs on protein quantification in the BCA assay. By combining the BCA assay with inductive coupled plasma mass spectrometry (ICPMS) for quantification of metal-based NPs, the amount of protein per NP can be validated.49 Furthermore, ICP-MS analysis of the S/Au ratio can help to quantify the absolute number of corona proteins per NP.49 Bettmer et al. used the alternate strategy of hard protein corona (BSA) isolation to quantify the S/Au ratio of Au NPs with their coronas. To account for the differing compositions of proteins, 39.6 S atoms per protein was used to determine the maximum amount of proteins adsorbed onto the different sizes of Au NPs. The protein number significantly decreased in the hard protein corona of Au NPs as the NP size increased. 1926


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Figure 3. Kinetics of formation of silica NPs−protein corona in static NP conditions. (a) 2D CLSM images of NP fluorescence intensity changes during the incubation. (b) Triplicate (normalized) results of NP fluorescence intensity (I) as a function of incubation time. (c) Linearized plots of data plotted in (b). (d) Enlarged version of linearized data (Phard1 and Phard2). (Adapted with permission from ref 55. Copyright 2019. American Chemical Society.)

the thickness of the protein corona to the plasma concentration has been observed. Weiss et al. monitored the time-evolution of protein corona formation by confocal laser scanning microscopy with microfluidics (CLSM), in which fluorescence intensity increases upon protein adsorption onto NP surfaces. Protein corona formation began after a millisecond of introducing the NP to the protein; the process completed after several minutes, at which point it reached an equilibrium. The authors divided protein corona formation into three phases. The first phase (P1hard) is the direct binding of proteins with high binding affinity onto the surface of NPs surface, forming a hard corona. The second phase (P2hard) is an irreversible protein−protein interaction, in which new proteins adsorb onto the surface of the hard corona. The third phase (Psoft) is a reversible process of the loose binding of proteins (outer layer; Figure 3). An enhancement of fluorescence intensity was observed with increasing incubation time; the protein corona formation reached a plateau at 190 ± 62 s after incubating silica particles with HSA-Cy5 proteins.55

coronas, due to the lack of appropriate techniques to assess the “hardness” of protein coronas. After washing protein-coated NPs several times in a protein-free buffer, the remaining protein layer is often assumed to be a hard corona.52 The thickness of a hard corona has been shown to be directly proportional to the concentration of proteins or/and incubation time with the protein solution.3 Most studies are thought to have examined the properties of hard coronas. Right now, we have very limited understanding of the biological behavior of soft coronas. Several studies have examined the thickness of soft coronas by comparing the full protein corona to the hard corona thickness. However, such an indirect method must be carefully evaluated to avoid variable results. Additionally, the comparison between ex vivo and in vivo measurements can only provide approximate results due to the difficulties in comparing measurements under different solute conditions.3 The differences between hard and soft coronas are not always sufficiently clear to identify and understand. Schaffler et al. proved the impossibility of measuring soft corona thickness by dynamic light scattering (DLS),53 while Walczyk et al. attempted to calculate the soft corona thickness using different methods (e.g., the sum of hard corona thickness subtracted from the full protein corona).47 DCS is one of the major methods to study the soft corona, but this technique has the limitations of mathematical modeling. For valid determination of size by DCS, the shape and distribution density of the NPs must be known.37,47 The “true” size of a NP−protein corona has been computed using a core−shell model of two densities (i.e., bare NPs and the protein adsorbed around the NPs). This is a simple model to analyze shell coated NPs, where the shell represents the protein corona thickness.54 This method has been commonly used for examining polystyrene NPs under different conditions. Remarkably, after the isolation step, no change in NP size has been reported. However, a directly proportional reduction in

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IDENTIFICATION OF PROTEIN CORONA COMPOSITION Generally, most of the methods used to identify coronal proteins are based on SDS-PAGE and mass spectrometry (MS). The protein composition of a corona can be determined in vitro after isolation of NP−protein coronas from a biological fluid. The gel electrophoresis method may be combined with MS,4,5,47 matrix-assisted laser desorption-ionization time-offlight MS (MALDI-TOF MS),46,56 and/or electrospray ionization MS (ESI-MS)49 for further detection of the electrophoretically resolved proteins by trypsin digestion and peptide analysis. Lundqvist et al. studied the size and surface charge effects of polystyrene NPs on protein corona formation in human plasma. An 80% homology was found in the coronal 1927


Review


Figure 4. Analytical techniques for analyzing the binding affinity and conformational changes of proteins adsorbed onto NPs. (a) ITC plot of 60 nm carboxylate-modified polystyrene NPs titrated with a BSA solution. Differential power of titration (top) and integrated heat of the BSA adsorbed on NPs (bottom). (Adapted with permission from ref 59. Copyright 2014. American Chemical Society.) (b) SPR analysis of the interactions of 60-fold diluted plasma over 70 nm 85:15 NIPAM/BAM(blue) or 50:50 NIPAM/BAM(red) particles for 30 min (left) and 24 h (right). (Adapted with permission from ref 4. Copyright 2007. National Academy of Sciences.) (c) DNA detection based on TREC-AFM imaging. Illustration of the detection of DNA binding to surface-immobilized target DNA (left). Recognition (dark spots) and topography images of singletarget DNA molecules (right). (Adapted with permission from ref 62. Copyright 2018. American Chemical Society.) (d) CD spectra Tf before and after interaction with pen-gold NPs reveal conformational changes. (e) Curve-fitted inverted second-derivative and curve-fitted inverted Fourier self-deconvolution of the amide I spectral region of Tf adsorbed on chiral surfaces of Au NPs. (Adapted with permission from ref 63. Copyright 2017. American Chemical Society.)

dihydrolipoic acid (end-functional group COOH)-modified QDs.46 Tenzer et al. studied different sizes of silica NPs and their protein corona complexes after interaction with plasma. The authors concluded that the composition of the protein corona formed around negatively charged silica NPs did not correlate with protein size or charge. Notably, the authors observed a size-dependent adsorption of prothrombin and thrombospondin-1 onto ∼55 nm silica NPs; these proteins were less adsorbed on ∼16 and ∼10 nm silica NPs.57

proteins around neutral polystyrene NPs. This was not affected by the different dimensions. Remarkably, immunoglobulin G (IgG) highly adsorbed onto the surface of 100-nm-diameter carboxyl modified NPs but did not efficiently bind to 50 nm NPs. The amino-modified 50 nm NPs adsorbed a larger number of apolipoproteins compared to the other size- and surface-modified NPs. The compositions of neutral charged NPs of different sizes were also compared. ApoB-100 was detected only with 100 nm NPs; ApoB-100 was not detected in the protein corona of 50 nm NPs. The authors hypothesized that 100 nm neutral-charged NPs preferentially interact with LDL, since ApoB is one of the main components in LDL.5 Wang et al. observed different corona compositions of surface ligands on solubilized QDs analyzed by gel electrophoresis and MS. This observation highlights the importance of NP modification in the formation of the protein corona. Immunoglobulin, HSA, and α2-macroglobulin were found in the protein corona of surface-modified CdSe/ZnS QDs. Interestingly, ApoA-1 has been observed in the protein corona of two types of QDs. Complement components (factor H and Cb3) have also been found on the most negatively charged

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MECHANISMS AND DETERMINANTS OF PROTEIN CORONA FORMATION Kinetics of Protein Binding on the Surface of NPs. Proteins constantly compete with each other for binding sites on the surface of NPs. Biomolecules with high affinity will replace more mobile biomolecules with low affinity to bind to the surface of particles, based on the Vroman effect.58 Thus, protein affinity plays a critical role in the formation of the protein corona. The adsorption affinity can predict the localization and composition of proteins on a NP’s surface as well as the formation of hard and soft protein coronas. 1928


Review

Bioconjugate Chemistry Isothermal titration calorimetry (ITC)59 (Figure 4a) and surface plasmon resonance (SPR)4,60 (Figure 4b) are techniques that can successfully assess the kinetics of protein adsorption. ITC is used to detect thermodynamic parameters of protein binding (ΔH, ΔS, ΔCp). The stoichiometry and Gibbs free energy are the results of protein−NP interactions. This technique provides different heating temperatures and various rates of combining NPs with protein. Ding et al. studied the interaction mechanisms between HSA and surfacemodified GO nanosheets (NSs) using ITC. This strategy revealed hydrogen bond-driven interactions of HSA with GO or GO−COOH, based on negative enthalpy (ΔH) results. Covalent bonds and hydrogen bonds were the main driving forces of GO−HSA interactions (due to the interactions between Lys and Arg on the surface of HSA with the epoxy groups on GO). The interaction between HSA and GO− COOH is driven through hydrogen bond interactions, due to the blockage of the epoxy groups on GO by the carboxyl groups. The ITC data from the interaction of HSA with GO− PEI or GO−CS presented a positive entropy (ΔS), indicating an interaction driven by hydrophobic forces. SPR data demonstrate stronger interactions between GO and HSA compared to surface-modified GO. The binding affinities of NSs during the interaction with HSA follow the order: GO > GO−COOH > GO−CS > GO−PEI.59 Fleischer et al. found that negatively charged NPs have a strong binding affinity for serum albumin compared to neutral and positively charged NPs.61 Their ITC results demonstrate that, while the ΔH of BSA binding to amine-modified and carboxylate-modified polystyrene NPs are similar, the number of adsorbed BSA molecules is ∼30-fold higher on COOH−NPs than that on NH2−NPs. Cederval et al. used ITC to calculate the degree of surface coverage of NPs treated with albumin. On average, each 70 nm NP bound 620 protein molecules, while 4650 protein molecules were calculated to be present on each 200 nm NP. This finding suggests that HSA preferentially adsorbs onto hydrophobic NPs with larger surface coverage. SPR experiments to investigate the 70 and 200 nm NPs interactions with HSA and fibrinogen (as a simple model) showed a fast dissociation event, suggesting that the interactions off these proteins with NPs are rapid. Notably, the authors observed a more rapid dissociation of HSA and fibrinogen from more hydrophobic NPs, compared to more hydrophilic particles.61 Ritz et al. investigated the interactions between human serum and polystyrene NPs with six different surface functionalization groups. The ITC data suggest that carboxyl- and sulfonatefunctionalized NPs interact more strongly with human serum compared to amino-functionalized polystyrene NPs.61 SPR can also provide real-time binding measurements, based on the changes of surface plasmon waves’ oscillation. NPs are attached to gold-modified microchips, and proteins are then injected to flow over the surface of the chips. Changes of the angular position of the SPR peaks may represent a NP− protein interaction. Chong et al. used this technique to examine the interactions between GO and plasma proteins. Based on their SPR data, the Kd values of binding to GO rank as follows: BFG > Ig > Tf > BSA. Bovine fibrinogen (BFG) showed the strongest binding affinity to GO.23 Oh et al. developed a new method for affinity studies based on a DNA detection topography and recognition (TREC)-AFM platform (Figure 4c), which provides a broad and rapid approach for alternative affinity label-free nanoscale biosensing. The authors characterized DNA arrays at the level of single DNA/DNA

hybridization events. Of note, faster recording times and higher pixilation density were observed, compared to force mapping. One of the advantages of this novel method is the ability to study the affinity with minute amounts of sample material.62 Determination of Protein Structures as Affected by the Physiochemical Properties of NPs. After interaction with a NP, the structure of protein may change. Since such an alteration can have a profound effect on protein function, it is vital to determine the structural changes affected by NPs. Circular dichroism (CD) is a widely used spectroscopic technique used to detect the conformation of proteins. CD spectra reveal the interactions of various chemical groups in the vicinity of optically active molecules (i.e., proteins and peptides) below 240 nm. The region 170−240 nm is typically very sensitive to peptide bond adsorption and conformational changes of proteins. Normalized background signal of NPs needed to remove the substrate from the protein−NP complex signal to avoid the possible impact of NPs. CD spectroscopy can be used to measure the interactions between NPs and proteins in real time and in the presence of unbound proteins.63 Using CD spectra, Ge et al. revealed interactions of CNTs with four blood proteins, including BFG, γ-globulin, transferrin, and BSA. After 10 min of interaction with CNTs, the secondary structure of all four proteins was significantly altered. β-Sheets are increased with a concomitant decrease in α-helical structure.24 Wang et al. investigated secondary structural changes in coronal BSA on the surface of Au NPs. The rapid decrease of α-helices through increasing β-sheet structures occurred through the transformation of protein disulfide bonds into Au−S coordination.64 Ding et al. found no apparent changes in the α-helical structure of HSA during interaction with four types of GO, using CD spectroscopy. The authors ranked the effects of GO NSs on HSA conformational changes in the following order: GO−PEI > GO > GO−CS > GO−COOH. As expected, the highest change in HSA conformation was the result of strong electrostatic and hydrophobic interactions with GO−PEI NSs.60 Another useful tool in the characterization of protein corona is synchrotron radiation CD (SR-CD; Figure 4d), which can enhance the accuracy of CD measurements.64 Using SR-CD, protein structure is assessed in the low UV regions to generate more comprehensive and accurate results.65 Leblanc et al. explored the possibility of using CD spectroscopy to determine protein concentration and the effects of carbon dots (∼4 nm), Au NPs (∼10 nm), and PEG on CD signals in protein−NP complex systems. Several NPs of different shape and size, along with human serum albumin (HSA), transferrin (Tf), and chicken ovalbumin (OVA), were examined. A drop in photon amount in the range of 200−190 nm in the CD spectra was shown due to the strong absorbance of NPs at a fixed concentration. The authors concluded that the NPs did not interfere with CD spectra until the photons could reach the measuring detector.66 Another technique, Fourier transform infrared (FTIR) spectroscopy (Figure 4e), can be used to confirm the structural changes in proteins, as induced by NPs, based on the adsorption peaks of amide vibrational bands. The main amide band is the amide I region located in the 1700−1600 cm−1 range.63,67 The FTIR data may reveal protein and NP aggregation after their interaction.68 Wang et al. found that the absorption of plasma proteins onto Au and Ag NPs was determined by fast electrostatic forces and slow covalent bonding. The secondary structures of non-thio-proteins did not change, in contrast to thio-proteins for which the 1929


Review


Figure 5. Characterization of colloidal stability of NPs after introducing with proteins through size distribution. (a) DLS measurements of various sizes of Au NPs before and after formation of the protein corona. (b) Measurements of mean diameters of left: (1) Au NPs (10 nm); (2) Au NPshard protein corona (16 nm); (3) Au NPs-hard protein corona and IgG antibody (16 nm); (4) Au NPs-hard protein corona and BSA-antibody (26 nm); right: (1) Au NPs (10 nm); (2) Au NPs-BSA (21 nm); (3) Au NPs-BSA and IgG antibody (22 nm); (4) Au NPs-BSA and BSA-antibody (37 nm). (Adapted with permission from ref 3. Copyright 2010. American Chemical Society.) (c) DCS results after 1 h introducing of 200 nm silica NPs with various concentrations of plasma proteins (top), and plasma concentration dependent NP−protein corona thickness distribution (bottom). (Adapted with permission from ref 37. Copyright 2011. American Chemical Society.)

vibrations in ultrasmall volumes and in ultrahigh resolution for further identification of samples. Nowadays, nano-FTIR is a viable technique to examine protein−NP interactions at very (ultra) small quantities and generate extraordinarily sensitive mapping of protein conformations at nanoresolution approaching single-protein sensitivity.69 Raman spectroscopy (RS)70,71 and surface-enhanced Raman scattering (SERS)71 are spectroscopic methods based on the vibrations of molecules. In the case of SERS, only proteins with Raman active molecules adsorbed onto metal NPs contribute to the signal. The Raman spectra of pure proteins come from aromatic side chains and peptide and sulfur side chains. Shashilov et al. characterized the conformation of amyloid fibrils by hydrogen−deuterium exchange using deep UV resonance RS.81 It was easier to assess protein−NP interactions in a liquid state, but solid state analysis was also possible. Zhang et al. examined the SERS spectra of lysozymeNPs. A RS of lysozyme and drop-dried lysozyme was generated to analyze the spectral changes of the α-helical to a β-sheet, or random coil, shift upon protein adsorption to the Au NP. RS and SERS features can also be applied to characterize the targeted environment of proteins. Tryptophan residue peaks were observed at 1360 or/and 1340 cm−1. More hydrophobic bonds were located at the tryptophan residues upon interaction with gold NPs. The possible covalent interactions

conformation changed during the absorption on Au or Ag NPs. Four plasma proteins were incubated with Au or Ag NPs for further investigation of the secondary structure changes by FTIR. The secondary structure of thiol-proteins changed in a time dependent manner, but non-thiol-proteins remained unchanged after incubation with Au and Ag NPs.68 Wang et al. studied the effect of NPs’ chiral surface on the conformation of the adsorbed proteins. Slight changes in the Tf secondary structure after adsorption onto a chiral surface of Au NPs were characterized using CD and FTIR (amide I, amide II, and amide III regions). A decrease in α-helical structure and increase in β-sheet content after Tf adsorption onto the chiral surfaces of Au NPs was observed.63 Zhang et al. studied the secondary structure of various proteins using a solid-film sampling method (FTIR). Proteins with primarily α-helical, βsheet, and mixed structures were chosen for secondary structure determination in amide regions. The authors concluded that protein concentrations must be at least 0.5 mg/mL for the determination of protein conformational changes using the solid-film sampling.78 Hillerbrand et al. demonstrated the extraordinary sensitivity of FTIR nanospectroscopy (nano-FTIR), which can generate an infrared adsorption spectrum at a resolution of 30 nm for a very small volume of ferritin (around 1 attogram). Nano-FTIR can be correlated to conventional FTIR, using databases of molecular 1930


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integrated proteins reduces nonspecific interactions and can play a vital role in corona formation. The presence of proteins on the leukosome’s surface also can promote the adsorption of specific proteins.76

(SS stretch region) during the interaction between lysozyme and Au NPs were observed in the 500−525 cm−1 region.71 Nuclear magnetic resonance (NMR) is a powerful technique that can reveal detailed information about the dynamics of NP−protein interactions, the structural changes of proteins, and the chemical nature of molecules in solution.72 The chemical shifts of bare NPs and native proteins are unique. A peak intensity change, peak shift, and/or appearance of a new peak(s) can indicate the adsorption of protein on the surface of NPs. NMR spectra can not only assess protein adsorption, but also distinguish newly formed chemical bonds in protein−NP complexes.72 In an NMR study of the dynamic equilibria of bound and unbound human carbonic anhydrase I (HCAI) and silica particles, it was found that particle curvature strongly influences bound protein secondary structure.52 The NMR data revealed a conformational change in the protein due to adsorption onto the particles, measured immediately after the interaction of HCAII with silica NPs. Long-term interactions exhibited a narrow shift, indicating a strong adsorption of protein on silica NPs as well as loss of proteins’ tertiary structure.72 Stayton et al. have evaluated the in situ secondary structure of peptides on hydroxyapatite surfaces.73 Lundqvist et al. analyzed the adsorption of HCA on silica NPs. Notably, a significant time-dependent change of the tertiary structure of HCA was detected in near-UV CD spectra. Conformational changes in the protein during the interaction with NPs were assessed by NMR, which revealed an adsorption of non-native conformational protein onto NPs with the appearance of new peaks of tertiary structure. The authors concluded that NP curvature has a strong influence on the perturbation of protein secondary structure. Fifteen nanometer silica NPs changed the secondary structure of the protein 6-fold more than 6 nm NPs.52 In addition to CD spectroscopy, classical UV−vis spectroscopy can be used as a powerful tool for studying conformation changes of biomolecules induced by NPs. Xu et al. showed that biomolecules adsorbed on magnetic Au NPs can be effectively released by adding NaBr. The authors used CD and UV spectroscopy to show that Mb and BSA proteins did not change in conformation after being releasing from the NPs.74

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EFFECTS OF PROTEIN CORONA ON THE PHYSIOCHEMICAL AND BIOTRANSFORMATIONAL PROPERTIES OF NANOPARTICLES Impact of the Protein Corona on the Physiochemical Properties of NPs. DLS (Figure 5a,b) is a widely used technique to determine the hydrophobic diameter (HD) of NPs and/or their complexes with protein. This technique can be used to distinguish changes in hydrodynamic NP diameter before and after protein corona formation. To derive reliable results from DLS, the NPs and proteins need to exhibit an approximately homogeneous shape in the dispersion. Research on the impact of various physicochemical properties on the size of the NP−protein corona has been performed.3,77 The addition of proteins to the surface of a NP will change the particle’s isoelectric point (pI) and zeta potential. Protein aggregation is also correlated with NP charge. After interacting with NPs, the pI of a protein will decrease, leading to NP aggregation.21,77 The number of adsorbed proteins on the surface of a NP correlates with NP charge.6 Interestingly, stronger interactions between negatively charged Au NPs and negatively charged proteins were observed compared to positively charged NPs.78 NP surface charge is another crucial factor in protein corona formation. Positively charged NPs preferentially adsorb proteins with pI 5.5, such as IgG.9 Gessner et al. observed an increase in the adsorption of plasma proteins on negatively charged NPs with increasing surface charge density.10 Au NPs with positive, negative, and neutral tails were used to detect protein denaturation in the presence of charged tails; the conformation of proteins was protected in the presence of neutral NPs, in contrast to positive or negative NPs.8 Goy-Lopez et al. investigated the interaction between HSA and Au NPs in time dependence experiments. The authors observed a long colloidal stability after introducing HSA to Au NPs, where the zeta potential of the protein−NP complexes was around −30 mV (which does not favor long-term conjugate aggregation). Importantly, there were no protein aggregates observed during the thermal process, which was confirmed by the DLS data (i.e., large-sized proteins were not detected).77 Chanana et al. studied the physicochemical properties of Au NPs coated with proteins and labeled with a fluorescent dye, as a model system for investigating the impact of cellular uptake on the colloidal stability of NPs. Protein-coated NPs were colloidal stable in solution with different salts and biomolecules (e.g., complete cell medium); however, the enzymatic degradation of the protein corona at pH 4.7 was observed, while the Au NPs were not affected by the degradation of the protein corona.79 Monopoli et al. examined plasma concentration-dependent hard corona formation around silica and polystyrene NPs using the core−shell model of DCS (Figure 5c).37 They had shown that hard corona thickness has significance in various plasma concentrations for better interpolations of in vitro and in vivo studies. Blundell et al. investigated time dependent carboxyl particle−protein corona formation by measuring zeta potential

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IN VIVO PROTEIN CORONA CHARACTERIZATION Research has unequivocally shown that the independent characteristics of NPs, such as size, chemical nature, surface charge, surface modifications, incubation conditions (like temperature and time), and the characteristics of biological fluids (e.g., concentration) all affect the formation of protein coronas around NPs. The features of protein coronas in vivo must also be understood. Several methods for investigating the corona in vivo have been described. Carril et al. presented a strategy in which NPs were labeled with 19F in the complex biological fluid and then examined by 19F diffusion-ordered NMR.75 This method is not optically based and can be used in various conditions to quantify protein adsorption on NPs in situ. The authors demonstrated that hydrodynamic radii changes in vivo can be indicative of NP behavior (e.g., NPs agglomerate to affect increasing hydrodynamic radii, NPs degrade and exhibit decreasing hydrodynamic radii, and protein adsorption leads to a small increase in NP size). Corbo et al. studied the temporal evolution of the protein corona in vivo. The integration of leukocyte membrane proteins into leukosome bilayers was affected by the number and type of adsorbed plasma proteins. The masking effect of 1931


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Figure 6. Characterization of “hard” and “soft” protein corona formation on NPs. (a) Illustration of hard and soft corona measurement during NP−protein interactions. In represents the positions of NPs after they translocate through the TPRS nanopore. T represents the duration (s) of NP translocation through the nanopore. (b) Schematic representation of NP−protein corona complexes: (i) NPs precoated with serum proteins, (ii) introduction of plasma proteins to the precoated NPs, (iii) displacement of hard corona proteins, and (iv) dissociation of proteins exhibiting loose protein−protein interactions. (c) Zeta potential measurements after 5, 10, 15, 20, 30, and 60 min incubation of NPs with plasma proteins. (Adapted with permission from ref 21 Copyright 2016. Springer.)

containing peptides and Ag NPs by forming Ag−S species, with further oxidation and formation of Ag−O species.81 The authors also investigated the possible adsorption mode of BSA on Au NPs through sulfur interactions by using XANES (Figure 7c−e). The interaction of BSA and Au NP was driven through Au−cysteine interactions, while Au NP did not form Au−S bonds with methionine. The XANES results showed that disulfide content decreased, by transforming disulfide bonds into Au−S, during the adsorption of the BSA onto the surface of gold NPs.64 Hu et al. investigated how pulmonary surfactants, as a part of biomolecular coronas formed in the lung, can impact Ag NP dissolution, followed by silver ion release and decrease of the pro-inflammatory potential of the NP in the lung. The influence on the inflammatory potential of Ag NPs was explained by the formation of a physiological core around NPs, preventing or enhancing aggregation of the NPs in biological environments.82 Control of NP Protein Corona Formation through Surface Modification. As discussed above, the formation of the corona is a dynamic process dependent on the physicochemical properties of NPs (i.e., size, charge, surface modification, and surface curvature) and the properties of the biological system (e.g., circulation time, speed, pressure). The important parameter, such as pH dependent hydrodynamic diameter change, is one of the major physiological properties of a model Au NPs for regulation of NP−protein interactions.83 In recent years, researchers have studied the effects of surface modification of NPs (e.g., PEG, PEEP, antibodies) on the properties of the protein corona. It is wellknown that polymers prolong the half-life of NPs in biosystems, though there are caveats to decorating the surface of NPs with polymers. Dai et al. discovered that modification of Au NPs with PEG reduced the formation of the protein corona, leading to a loss of targeting ability.84 The authors

changes with tunable resistive pulse sensing (TRPS). In this technique, the electrophoretic mobility of each individual particle is measured under the various incubation time points by passing through the TRPS nanopores. The conical sensing zone and blockade events at various positions (I) and blockade duration times (T) as the particles pass through the nanopore are presented in Figure 6a. The zeta potential of carboxyl particles precoated with serum proteins after introducing plasma proteins (for 5 min) was measured as well. The zeta potential of the particles after a 5−10 min (i−ii) incubation with plasma decreased due to the interactions of plasma proteins with the newly introduced particles were shown in Figure 6b,c. After 15 min (iii) of incubation, the zeta potential reached its lowest value during the experiments (reversible protein adsorption). After 20 min and then gradually to 60 min (iv), the zeta potential of the particles became more negative, showing enhanced particle translocation velocity through the pore.21 Interestingly, all modified NPs developed an organic coating under biological conditions via protein adsorption. The bare NPs rapidly agglomerated after interacting with biological fluids. In view of this, colloidal stability is a critical parameter, which may impact cytotoxicity and the immunological effects of NPs. Impact of Protein Corona on NP Biotransformation. Sutherland et al. reported an important role of soft corona formation in the sulfidation of Ag NPs in vitro. Theirs was the first report of a functional effect of soft corona proteins with a decreased amount of silver in nano-Ag2S. Importantly, the soft corona proteins impacted the transport of Ag+ by increasing sulfide formation. As previously shown, the formation of sulfide can decrease the toxicity of Ag NPs.80 Wang et al. investigated the possible chemical transformation of Ag NPs during cellular uptake for potential medical use by SR-X-ray absorption near edge structure (SR-XANES) spectroscopy (Figure 7a,b). The authors observed possible interactions of cysteine or cysteine1932


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Figure 7. Characterization of NP degradation after interaction with extracellular proteins and then cellular uptake. (a) Chemical species of silver NPs determined by K-edge XANES. (b) Changes in the silver chemical species of Ag NPs after THP-1 cellular uptake according to XANES results. (c) Representative TEM image of the interaction between Au nanorods and sulfur-containing molecules (BSA, cysteine, cystine, methionine). (d) Detection of sulfur species changes in cysteine, methionine, and cystine after interaction with Au nanorods. (e) Composition of sulfur chemical species in BSA and the BSA corona on Au nanorods. (Adapted with permission from refs 81, 64. Copyright 2015 and 2013 American Chemical Society.)

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CONCLUSION AND FUTURE PERSPECTIVES The rapid interaction of proteins and NPs will immediately form a soft corona on the nanosurface, which is then slowly replaced by a hard corona (in the scale of minutes to hours). Previous studies have shown the formation of multiple layer protein coronas on the surface of NPs.86 There are still several challenges to overcome in this field: 1. Which proteins in the protein corona are vital for the physiological behavior of NPs? What is the main component of the protein corona responsible for a NP’s physiological behavior? 2. Can the protein corona components control or predict further interactions with the organisms? 3. Does the soft corona play a crucial role under genuine biological conditions? 4. Are the current techniques sufficient for monitoring protein corona formation in vivo and ex vivo? As discussed above, the protein corona is a type of fingerprint of an NP in a biosystem. A single protein on the NP surface can stimulate a unique biological or immunological response. There remains a lot to learn on how to control (or prevent) protein corona formation through size, surface, or shape modification of nanomedicines or by precoating NPs with specific proteins for targeting purposes. Also, we may use the protein corona fingerprints of specific diseases to more deeply understand disease pathogenesis and/or sense disease

suggested that the length of the PEG core must be less than the length of linkers or ligands for successful recognition by cell receptors. Furthermore, when hydrophilic polymers are coated with proteins, an immune response can be triggered, increasing the in vivo stability of the medicine or nanocarrier (known as the “stealth effect”). Wurm et al. investigated the stealth effect using PEGylated and PEEPylated polystyrene nanocarriers.85 The authors showed that after polymer coating, the protein corona formation decreased, with the exception of nonspecific plasma proteins. Further investigation showed that Apo J (a.k.a. clusterin) precoated nanocarriers, together with a polymer core, reduced protein adsorption and decreased nonspecific cellular uptake. Another interesting approach in control of precoating the protein corona with antibodies. Tonigold et al. examined the effects of precoating polymer-modified polystyrene NPs40 with an antibody. Antibody precoated NPs were not completely covered by a protein corona. Furthermore, serum concentration did not affect the targeting properties or cellular uptake of antibody precoated NPs. The study showed the importance of controlling the formation of the protein corona for successful nanomedicine development. The stealth effect is not only a polymer core-dependent event, but is followed by the adsorption of specific proteins. The specific protein adsorbed may be controlled by the presence of antibodies on the surface of nanocarriers. 1933


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Bioconjugate Chemistry in the early stages. The burgeoning research in this field also provides new perspectives for designing highly efficient NPbased nanomedicine or nanocarriers for personalized drug delivery.14,87 Creating novel methodologies and analytical techniques to study protein−nanoparticle interactions, as well as the rates of association and dissociation, as a function of protein and particle type in different physiological conditions is essential for a deep understanding of the biological behavior of a nanomedicine under physiological or pathophysiological conditions. To predict the in vivo biological behavior of a protein corona coated NP as a nanomedicine, a complete understanding of NP−protein interactions in biological or biomimetic conditions must be further studied.

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SPR, surface plasmon resonance; NSs, nanosheets; BFG, bovine fibrinogen; TREC, topography and recognition; CD, circular dichroism; SR-CD, synchrotron radiation CD; Tf, transferrin; OVA, chicken ovalbumin; FTIR, Fourier transform infrared; RS, Raman spectroscopy; SERS, surface enhance Raman scattering; NMR, nuclear magnetic resonance; HCA, human carbonic anhydrase; HD, hydrophobic diameter; pI, isoelectric points; TRPS, tunable resistive pulse sensing; SRXANES, SR-X-ray absorption near edge structure spectroscopy

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Didar Baimanov: 0000-0002-5805-3303 Rong Cai: 0000-0001-6793-5765 Chunying Chen: 0000-0002-6027-0315 Author Contributions ‡

The manuscript has been written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Didar Baimanov and Rong Cai contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by Ministry of Science and Technology of China (2016YFA0201600 and 2016YFE0133100), the Science Fund for Creative Research Groups of the National Natural Science Foundation of China (11621505), the National Natural Science Foundation of China (31700879), the CAS Key Research Program for Frontier Sciences (QYZDJ-SSW-SLH022), the CAS interdisciplinary innovation team, and the National Science Fund for Distinguished Young Scholars (11425520). D.B. appreciated the financial support from the CAS-TWAS President’s Fellowship.

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ABBREVIATIONS NPs, nanoparticles; AFM, atomic force microscopy; TEM, transmission electron microscopy; SEM, scanning electron microscopy; GO, graphene oxide; rGO, reduced GO; BSA, bovine serum albumin; CNTs, carbon nanotubes; HSA, human serum albumin; FS, fluorescence labeled technique; FCS, fluorescence correlation spectroscopy; QDs, quantum dots; DCS, differential centrifugal sedimentation; BCA, bicinchoninic acid; ICP-MS, inductive coupled plasma mass spectrometry; QCM, quartz crystal microbalance; CytC, cytochrome C; Mb, myoglobin; QCM-D, quartz crystal microbalance measurements with dissipation; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; DLS, dynamic light scattering; CLSM, confocal laser scanning micros-copy with microfluidics; MS, mass spectrometry; Apo, apolipoprotein; MALDI-TOF MS, matrix assisted laser desorption-ionization time-of-flight MS; ESI-MS, electrospray ionization MS; ESIMS/MS, electrospray ionization-tandem mass spectrometry; IgG, immunoglobulin G; ITC, isothermal titration calorimetry; 1934


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