Nanoparticles present in any biological environment are exposed to extracellular proteins. These proteins adsorb on the surface of the nanoparticle forming a “protein corona.” These proteins control the interaction of nanoparticles with cells. The interaction of proteins with the nanoparticle surface is governed by physical chemistry. Understanding this process requires spectroscopy, microscopy, and computational tools that are familiar to physical chemists. This perspective provides an overview of the protein corona along with two future directions: first, the need for new computational approaches, including machine learning, to predict corona formation and second, the extension of protein corona studies to more complex environments ranging from lung fluids to waste water treatment.

Nanoparticles (NPs) present in any biological environment are exposed to a complex mixture of proteins. A subset of these proteins adsorb on the surface of the NP, forming a “protein corona” (Fig. 1).1–10 These adsorbed proteins, rather than the pristine NP, dictate the subsequent interaction of NPs with cellular receptors. Corona formation is a physical chemistry question. The process is governed by kinetics and thermodynamics with more abundant proteins forming the initial corona and proteins that interact more strongly with the NP surface forming a long-lived “hard” corona.12–18 The interaction of the protein with the NP surface depends on a combination of hydrophobicity and hydrophilicity, van der Waals forces, hydrogen bonding, and, in the case of gold NPs and thiol-containing proteins,19–22 possibly gold-thiol bonds.

FIG. 1.

The formation of a protein corona is governed by the kinetics (k) and thermodynamics (K) of the interaction of proteins with the NP surface. Representative blood serum proteins are shown: serum albumin (SA), immunoglobulin G1 (IgG1), alpha-2 macroglobulin (α-2 mg), and apolipoprotein A-1 (apo1 A-1). The protein corona is classified as a “hard” corona of tightly bound proteins and a “soft” corona of weakly bound proteins. Although typically shown in schematics as a layered corona, the actual corona is not well-structured.11 Modified with permission from Runa et al., J. Phys. Chem. B 121, 8619 (2017). Copyright 2013 John Wiley and Sons. Reprinted with permission from C. C. Fleischer and C. K. Payne, Acc. Chem. Res. 47, 2651–2659 (2014). Copyright 2014 American Chemical Society.

FIG. 1.

The formation of a protein corona is governed by the kinetics (k) and thermodynamics (K) of the interaction of proteins with the NP surface. Representative blood serum proteins are shown: serum albumin (SA), immunoglobulin G1 (IgG1), alpha-2 macroglobulin (α-2 mg), and apolipoprotein A-1 (apo1 A-1). The protein corona is classified as a “hard” corona of tightly bound proteins and a “soft” corona of weakly bound proteins. Although typically shown in schematics as a layered corona, the actual corona is not well-structured.11 Modified with permission from Runa et al., J. Phys. Chem. B 121, 8619 (2017). Copyright 2013 John Wiley and Sons. Reprinted with permission from C. C. Fleischer and C. K. Payne, Acc. Chem. Res. 47, 2651–2659 (2014). Copyright 2014 American Chemical Society.

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The phrase “protein corona” is, in some sense, a rebranding of our immune system’s opsonization process. The immune system must recognize and destroy unwanted biological materials such as pathogens and dead cells.23–25 An opsonin is a molecule that marks this cargo for uptake and degradation by specialized phagocytic cells. The same process, involving many of the same proteins, occurs with NPs or other synthetic materials.26,27 The success of our immune system leads to a major challenge for nanomedicine: NPs are cleared to the liver or spleen before they can reach their therapeutic target. Small NPs (<6 nm) are cleared to the kidneys.28 Early work in NP-based drug delivery recognized that decreased opsonization was essential in avoiding the clearance of NPs by phagocytic cells.29,30 A recent analysis of cancer nanomedicines estimates that only 0.7% of tumor-targeted NPs injected into the bloodstream reach tumors. The majority are instead cleared from circulation by phagocytic cells leading to accumulation in the liver and spleen.31 Even for NPs that do circulate through the bloodstream for a sufficient time to reach their target, there is the concern that corona proteins lead to mistargeting of cell surface receptors or masking of targeting ligands.32–36 

A description of the protein corona begins with the concentration and composition of the adsorbed proteins. In addition, and perhaps most relevant to physical chemists, the interaction of the proteins with the NP surface must also be considered. Studies of proteins interacting with planar surfaces have shown that this interaction often leads to changes in protein secondary structure, essentially some level of denaturation.37,38 Proteins can also undergo structural changes following interaction with NP surfaces dependent on NP diameter, surface functionalization, and specific corona protein.39–46 For example, circular dichroism (CD) spectroscopy of chymotrypsin and cytochrome c incubated with gold NPs (7 nm) showed that the structure of chymotrypsin was disrupted, but cytochrome c retained its secondary structure.47 Our group used a combination of CD spectroscopy, fluorescence spectroscopy, calorimetry, and fluorescence microscopy to show that adsorption of bovine serum albumin on the surface of a NP changes the structure of albumin, and this change in structure redirects the protein-NP complex to a different cell surface receptor (Fig. 2).7,33,34 In addition, the orientation of individual proteins on the NP surface must be considered as different regions of a protein will interact differently with cell surface receptors.48,49

FIG. 2.

Schematic illustrating how changes in the secondary structure of albumin, resulting from interaction with NPs, lead to binding of the protein-NP complexes to different cell surface receptors. Albumin adsorbed on the surface of anionic polystyrene NPs retains its secondary structure, and the albumin-NP complex binds to albumin receptors. Albumin adsorbed on the surface of cationic polystyrene NPs undergoes a change in secondary structure leading to binding to scavenger receptors. Reprinted with permission from C. C. Fleischer and C. K. Payne, J. Phys. Chem. B 118, 14017 (2014). Copyright 2014 American Chemical Society.

FIG. 2.

Schematic illustrating how changes in the secondary structure of albumin, resulting from interaction with NPs, lead to binding of the protein-NP complexes to different cell surface receptors. Albumin adsorbed on the surface of anionic polystyrene NPs retains its secondary structure, and the albumin-NP complex binds to albumin receptors. Albumin adsorbed on the surface of cationic polystyrene NPs undergoes a change in secondary structure leading to binding to scavenger receptors. Reprinted with permission from C. C. Fleischer and C. K. Payne, J. Phys. Chem. B 118, 14017 (2014). Copyright 2014 American Chemical Society.

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Motivated by applications in nanomedicine, many of which would require the intravenous introduction of NPs to the body, the majority of protein corona studies have focused on the interaction of NPs with blood serum proteins. Serum is the collection of proteins obtained from whole blood following the removal of cells and clotting factors. It is a mixture of hundreds of different proteins. The most abundant, by far, is serum albumin (55%).50–52 NPs injected into the bloodstream encounter a concentration of 35–50 mg/ml of albumin.50 Cells grown in culture, rather than in an animal, require serum proteins as a nutrient source. Different cell lines require different types of serum proteins, but many use fetal bovine serum at a 10% (volume:volume) concentration. Fetal bovine serum from commercial suppliers has a protein concentration of 30–45 mg/ml, meaning that NPs used in cellular-level experiments encounter 3–4.5 mg/ml of protein, the majority of which is bovine serum albumin. Relating experiments in cultured cells (in vitro) to animals (in vivo) faces the challenge of two obvious differences. First, NPs in vivo will interact with blood, rather than just serum. These in vivo NPs will encounter ∼10× greater concentrations of serum proteins than used in cell culture, clotting factors, blood cells, and the shear forces associated with circulation. Second, most cell culture experiments use bovine serum before progressing to in vivo experiments in mice with the hope of one day being used in humans, meaning that the serum proteins of at least three distinct species must be considered.

The differences between a corona formed in plasma, which contains clotting factors, and the one formed in serum have been investigated.53,54 Silica NPs (100 nm) were found to adsorb more proteins in human plasma [30 ± 3 µg protein/mg NPs by bicinchoninic (BCA) protein assay], compared to human serum (21 ± 2 µg protein/mg NPs) at equal protein incubation concentrations, with differing types of proteins enriched on the NP surface.53 For example, the protein corona of NPs incubated with serum was dominated by lipoproteins (93% of the relative weight). Coagulation proteins were the next most abundant protein type in the serum corona but at much lower levels (3%). In comparison, the plasma corona had a more varied distribution of proteins (e.g., 58% lipoproteins and 22% coagulation proteins). The plasma corona also resulted in greater uptake by macrophage cells, suggesting a greater clearance rate than would be predicted from experiments using serum. As an even more direct measure, the corona formed on polyvinyl alcohol-functionalized iron oxide NPs (90 nm) incubated for 15 min in rat serum was compared to the corona formed following 10 min of circulation through the rat circulatory system.54 As the authors note, this experimental design ignores the protein corona of NPs cleared from circulation during this time period, which would not be collected for analysis. Our own work with polystyrene NPs (200 nm) has shown that the protein corona formed at shear flow rates that mimic the circulatory system has a higher protein concentration and is enriched in plasminogen in comparison with the incubation conditions used in many corona experiments.55 Polystyrene NPs (200 nm) in a solution of serum proteins were subjected to flow with flow rates chosen to model capillaries (0.085 cm/s or 0.1 dyn/cm2) and veins (0.85 cm/s or 1.0 dyn/cm2).55–57 The protein-NP complexes formed following flow show decreased cellular binding, measured with flow cytometry. Our studies used polystyrene NPs, but the result appears general. Gold NPs (13 nm) functionalized with poly(ethyleneglycol) (PEG) or tannic acid also showed an increase in protein concentration in response to flow.58 Similarly, lipid NPs (140 nm), both unmodified and PEGylated, were found to have increased protein coronas in response to flow.59 

In addition to a comparison of in vitro and in vivo conditions, the other immediate question that arises is the relevance of experiments in fetal bovine serum, a standard nutrient source for cells in culture to, ultimately, applications in human nanomedicine. Researchers have compared the protein corona formed from mouse plasma vs human plasma and bovine vs human serum.60,61 A comparison of the fetal bovine serum and human serum corona formed on silica NPs (50 nm) as a function of a functional group [–NH2, –SH, and –polyvinylpyrrolidone (PVP)] and time (5 min–1 week) showed that albumin was the most abundant corona protein in all cases, but there were interesting differences in some of the less abundant proteins.60 For example, apolipoprotein B-100 was found in the corona of all NPs incubated with human serum but not fetal bovine serum.

Because corona proteins lead to clearance from circulation in the bloodstream, much work in the nanomedicine community has focused on reducing the protein corona, typically with poly(ethyleneglycol) (PEG). PEG is currently used to increase circulation times for lipid-based nanomedicines used in the clinic (e.g., Doxil, Neulasta, PEG-Intron, and Oncaspar) and for gold NPs in development (Aurimune).62,63 PEG does reduce the protein corona and increase circulation times,64–69 but small amounts of corona proteins remain.30,68,70–72 These residual corona proteins ultimately lead to clearance to the liver and spleen.5 Beyond PEG, zwitterionic polymers have shown promise for corona reduction.73–77 

Considering the difficulties in complete corona inhibition, recent research has taken a “if you cannot beat them, join them” approach, designing NPs to select for specific serum proteins on the surface.78,79 For example, covalent attachment of serum apolipoproteins and transferrin has been used to increase NP delivery to the brain,80–83 and to increase uptake into prostate cancer cells with upregulated receptors.16 A similar strategy for increasing nanomedicine circulation times is the design of NPs that avoid the adsorption of the specific proteins that initiate clearance from circulation, such as the complement proteins. Complement C3, an important immune system protein, is typically enriched in the protein corona. It is one of the three most abundant corona proteins on TiO2, polystyrene, iron oxide, gold, and silica NPs,71,84–86 despite being the 8th most abundant protein in serum.87 Previous work with PEGylated gold NPs (15–90 nm) showed that PEGylation reduced the adsorption of complement C3 and that these NPs had a decreased interaction with macrophage cells, suggested to be due to the reduced adsorption of complement C3.71 

This section outlines two major challenges in protein corona research: (1) We still lack the ability to predict what proteins will adsorb on a specific NP surface and if these proteins will undergo a structural change and (2) any molecule that can interact with a surface can form a corona, meaning that corona formation is much broader than just proteins and must be considered in environmental applications as well.

The formation of a protein corona is sometimes referred to as nonspecific adsorption, but in practice the proteins that interact with any surface are highly specific to that surface. These molecular details are what make the protein corona interesting to chemists. Currently lacking is an ability to predict which proteins will adsorb onto the surface of a particular NP. It is this area that both needs the most assistance and is perhaps most aligned with the expertise of physical chemists. In practice, the lack of predictive ability means that for each new NP and each new surface functionalization an experiment must be done to identify the concentration and composition of corona proteins. These experiments are fairly time and resource intensive. A colorimetric BCA protein assay (∼2 h) is used to measure protein concentration. Gel electrophoresis (∼3 h) provides an image of proteins separated by molecular weight. Western blotting (∼2 days), using an antibody, is necessary to truly identify proteins separated by electrophoresis. Gel electrophoresis and western blotting are useful for small numbers of proteins of interest with known molecular weights. Obtaining a complete list of corona proteins and their relative abundance requires the added time and expense of proteomics. All of these experiments require extensive controls to ensure that the NP or NP-biomolecule interaction does not introduce errors.88,89

Much previous work, including our own,33,34,90,91 has sought to identify trends in corona formation using small (∼2–5) numbers of NPs with varied core composition, diameter, or ligand. There has been extensive interest in correlating the effective surface charge of NPs with the resulting protein corona. There is an intuitive feel, proven incorrect in multiple investigations,33,86 that anionic NPs should have a corona dominated by cationic proteins and vice versa. Instead, previous work has shown little correlation with protein charge likely because proteins have regions of both positive and negative charge and because the corona interaction is not purely electrostatic. For example, a study of 11 different NPs (silica: 30 nm, 130 nm, and 140 nm and polystyrene: 115 nm; carboxylate- and amine-modified) with zeta potentials from −55 mV to +51 mV showed no correlation between the zeta potential and the protein isoelectric point.86 

The best attempt to correlate NP properties with the resulting protein corona comes from Walkey and co-workers who used the protein corona to predict cellular binding and uptake in human lung cells (A549).85 These experiments used a library of 105 gold NPs synthesized combinatorially using 3 different gold cores (15 nm, 30 nm, and 60 nm) and 67 different ligands (small molecules, polymers, peptides, and surfactants). The NP functionalization required 10 different synthetic protocols. Each of the 105 NPs was then analyzed individually including NP characterization (dynamic light scattering, zeta potential, transmission electron microscopy, absorbance, BCA protein assay, and gel electrophoresis) and proteomics [liquid chromatography with tandem mass spectrometry (LC-MS/MS)]. These experiments, which focused on the cellular interaction, showed that the protein corona was a predictor of cellular association and pointed toward the importance of hyaluronan-binding proteins, which are a major component of the extracellular matrix. While the focus of these experiments was the NP-cell interaction, the data obtained were also used to examine some correlations in adsorbed proteins. For example, the protein coronas formed on 15 nm, 30 nm, and 60 nm gold NPs with identical ligands were examined showing a wide range (∼40%–>80%) of overlap depending on the surface ligand.

These were Herculean experiments, partly due to the synthesis and characterization of the 105 NPs (3 gold cores, 67 ligands, and 10 synthetic protocols), but also due to the work involved in forming and isolating the protein corona. For corona experiments, NPs are incubated with serum proteins for a given time, the protein-NP mixture is then centrifuged to pellet the NPs with the associated hard corona, the supernatant containing unbound and weakly bound proteins is removed, and the hard corona-NP pellet is resuspended. This process is repeated until no unbound protein remains in the supernatant, determined by protein assay (BCA). Typically, 3–5 centrifugation steps are necessary to remove the unbound and weakly bound proteins. The hard corona is then digested off of the NP surface for proteomic analysis.

Ideally, a computational approach that reveals molecular-level details could be used in place of these extensive experiments.92–102 Computational approaches would be especially useful in predicting changes in protein structure resulting from the interaction of the protein with the NP surface as these experiments, typically CD spectroscopy, are especially time consuming. For example, Li and co-workers modeled apolipoprotein A-1 adsorbed on the surface of silver NPs (30 nm) with good agreement between simulations and CD spectra.97 Brooks and co-workers examined adsorption of a 56-residue model protein onto a NP surface as a function of NP diameter (6 nm, 20 nm, 80 nm, and flat) and temperature.95 These simulations, in agreement with CD and fluorescence spectroscopy experiments, showed that the native, folded, protein structure was preserved on small, hydrophilic NP surfaces. Increased hydrophobicity led to unfolding of the protein. Recent work has focused on atomistic detail in protein-NP simulations.99,103–105 For example, Liang and co-workers have carried out a comparison of density functional theory, quantum mechanical/molecular mechanics, and molecular mechanics simulations of a small alkyl amine model ligand on a gold surface.103 A recent review of computational approaches for modeling the protein corona provides a good overview of methods and challenges (Fig. 3).98 

FIG. 3.

Graph illustrating the size, in number of atoms, and time scale of current quantum mechanical (QM), molecular dynamics (MD), and coarse-grained simulation methods. The questions associated with protein corona formation include assembly, allosteric transitions, and protein folding, most compatible with Go-type MD simulations. From Li, Stevens, and Cho, Modeling, Methodologies and Tools for Molecular and Nano-Scale Communications: Modeling, Methodologies and Tools. Copyright 2017 Springer Nature. Reprinted with permission from Springer Nature.

FIG. 3.

Graph illustrating the size, in number of atoms, and time scale of current quantum mechanical (QM), molecular dynamics (MD), and coarse-grained simulation methods. The questions associated with protein corona formation include assembly, allosteric transitions, and protein folding, most compatible with Go-type MD simulations. From Li, Stevens, and Cho, Modeling, Methodologies and Tools for Molecular and Nano-Scale Communications: Modeling, Methodologies and Tools. Copyright 2017 Springer Nature. Reprinted with permission from Springer Nature.

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Any simulation method will struggle with the size of the protein-NP systems. In that sense, machine learning may provide a more feasible approach to protein corona prediction. Wheeler and co-workers have provided the best demonstration of this approach using random forest classification to determine what protein and NP parameters are important for corona formation [Fig. 4(a)].106 Their work with silver NPs (10 nm and 100 nm) shows that a combination of multiple protein and NP parameters factors into corona prediction [Fig. 4(b)].

FIG. 4.

Overview of a machine learning approach to protein corona formation. (a) Graphical depiction of the machine learning pipeline describing the operations used to produce a predictive model. Data acquired by proteomics (LC-MS/MS) were normalized, and non-numerical values were replaced with mean values during the preprocessing step. Grid search was then employed to minimize the generalization error of the model, and recursive feature elimination with cross-validation was then carried out to optimize the dimensionality of the database. Grid search was then employed again to reduce the generalization error on the optimized database. The model was then run and validated 50 times on randomly selected stratified database partitions to produce performance metrics. (b) Weighted importance of each feature included in the final model. Protein features are shown in green, NP features are shown in blue, and solvent features are shown in red. Error bars are shown with black lines. Reprinted with permission from Findlay et al., Environ. Sci.: Nano 5, 64 (2018). Copyright 2018, Author(s), licensed under a Creative Commons Attribution 3.0 Unported License.

FIG. 4.

Overview of a machine learning approach to protein corona formation. (a) Graphical depiction of the machine learning pipeline describing the operations used to produce a predictive model. Data acquired by proteomics (LC-MS/MS) were normalized, and non-numerical values were replaced with mean values during the preprocessing step. Grid search was then employed to minimize the generalization error of the model, and recursive feature elimination with cross-validation was then carried out to optimize the dimensionality of the database. Grid search was then employed again to reduce the generalization error on the optimized database. The model was then run and validated 50 times on randomly selected stratified database partitions to produce performance metrics. (b) Weighted importance of each feature included in the final model. Protein features are shown in green, NP features are shown in blue, and solvent features are shown in red. Error bars are shown with black lines. Reprinted with permission from Findlay et al., Environ. Sci.: Nano 5, 64 (2018). Copyright 2018, Author(s), licensed under a Creative Commons Attribution 3.0 Unported License.

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The majority of research in the protein corona community focuses on just that, the protein corona. But the interaction of a NP with the local environment is much broader. For example, lipids are also present in biological environments and will also adsorb on the surface of NPs forming a lipid corona.107,108 This is especially important for NPs that are inhaled, such as in a manufacturing setting, leading to a corona formed from the complex fluid of the lung with its combination of proteins and lipids.109,110

Even more broadly, NPs that are released into the environment may come into contact with soil or water sources.111,112 For example, silver NPs used as antimicrobials in consumer products are released into the sewer system.113 Emerging uses of NPs in agriculture as fertilizers, pesticides, and sensors directly introduce NPs to the environment.114–117 Molecular studies of environmental coronas have focused on the interaction of NPs with natural organic matter.118,119 For example, infrared spectroscopy has been used to compare the adsorption of humic acid on TiO2 and α-Fe2O3 NPs showing spectral differences as a function of NP composition.118 Humic acid and BSA coexist on the surface of TiO2 NPs,120 suggesting that both would be present during a human exposure.

It is now well-accepted that the protein corona is a key factor in determining the interaction of NPs with cells. A complete description of the protein corona includes the concentration of adsorbed proteins, composition of the proteins, and an examination of possible structural changes in response to the interaction of proteins with the NP surface. All of these factors are sensitive to differences in plasma, serum, species, and circulation. Beyond working to understand the formation of the corona, research, especially for nanomedicine applications, takes two approaches: (1) continued investigations of surface functionalizations to reduce corona formation, building on a long history of PEGylation and (2) a newer approach of designing a NP surface to select for certain serum proteins. Moving forward, two challenges are outlined in future directions: (1) we still lack the ability to predict what proteins will interact with a specific NP and (2) exploring the corona beyond proteins. The importance of the protein corona in nanomedicine and the environment makes it an exciting research area that uses concepts of fundamental physical chemistry in both experimental and computational approaches.

The author would like to thank the Payne Lab protein corona team at Georgia Tech and Duke and funding support from NIH, NSF, and the HERCULES: Exposome Research Center at Emory University’s Rollins School of Public Health.

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