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A Decade of the Protein Corona

Pu Chun Ke,† Sijie Lin,∆ Wolfgang J. Parak,¶ Thomas P. Davis*,†,‡ and Frank Caruso*,§

†ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia

∆College of Environmental Science and Engineering, State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, 1239 Siping Road, Shanghai 200092, China

¶Fachbereich Physik und Chemie and CHyN, University of Hamburg, 22607 Hamburg, Germany

‡Department of Chemistry, University of Warwick, Gibbet Hill, Coventry, CV4 7AL, United Kingdom

§ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, and the Department of Chemical Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia

ABSTRACT

In this Perspective, we reflect on a decade of research on the protein corona and contemplate its broad implications for future science and engineering at the bio–nano interface. Specifically, we focus on the physical origin and time evolution of the protein corona, differences in the nanoparticle–protein entity in vitro and in vivo environments, the role of stealth polymers to minimize the formation of the protein corona, relevant computational and theoretical developments, and the “biocorona”, a concept extrapolated from the field of nanomedicine. We conclude the Perspective by outlining future directions and opportunities concerning the protein corona in the coming decade.

THE PROTEIN CORONA – A BRIEF HISTORY

A decade has passed since the inception of the protein corona, as coined by Dawson, Linse and coworkers.1 Although the adsorption of proteins onto surfaces, including particles, has been long known, its impact on bio-nano science was not fully considered prior to that report. The concept of the protein corona drew an analogy between the aura of plasma surrounding the sun and the protein layers adsorbed on nanoparticles in a biological milieu. But the similarities between the stellar and nanoscale phenomena end there. Over the past decade, we have learned that the protein corona owes its presence to thermodynamics in an aqueous environment, especially to the minimization of free enthalpy, and is mediated by Coulombic and van der Waals forces, hydrogen bonding, as well as hydrophobic interactions. Nanoparticles are often engineered with specific surface chemistries. However, such surfaces only exist transiently when exposed to biological environments, which consist of a myriad of biomolecules. The nanoparticle–protein corona challenges our understanding and exploitation of the nanoparticle “core” designed for sensing and nanomedicine.2

To commemorate a decade of the protein corona, herein we reflect on the development of the concept beyond the scope of nanomedicine and contemplate its implications for future science and technology at the bio–nano interface.

THE PROTEIN CORONA – “SOFT” VERSUS “HARD”, IN VITRO VERSUS IN VIVO

Over the past decade, there has been intense focus on elucidating the physicochemical and biological identities of protein coronas, employing a range of concepts and methodologies from colloidal science, biochemistry, biophysics, and toxicology. A major driver for these research efforts originates from the field of nanomedicine, where nanoparticles are designed for diagnostics, targeting, and drug delivery. Upon introduction into a biological fluid, a nanoparticle first assumes a transient or “soft” corona rendered by proteins of high abundance and is subsequently coated over time by a “hard” corona3 or proteins of high affinity according to the Vroman effect.4 The formation of the protein corona reduces free enthalpy. First, upon protein binding, which is energetically favored, enthalpy is reduced. Secondly, upon protein binding, the hydration layer around the nanoparticles is displaced, which increases entropy. The consequences of such bio–nano interactions are multifold: first, they may increase the nanoparticle solubility and hence availability in aqueous environments; secondly, they may trigger the biophysical processes of protein misfolding and aggregation, while the incurred protein conformational changes may elicit an immune response from the host to eliminate the nanoparticle in circulation; lastly, but perhaps most importantly, they may mask the chemical or biological functionalities imparted to the nanoparticle in the laboratory (Figure 1).

Figure 1. A nanoparticle gains a new biological identity upon its dynamic interactions with biological fluids, giving rise to a protein corona (shown as adsorbed green, blue, and cyan globules), which consequently influences drug delivery and targeting of the functionalized (illustrated as aqua blue fibrils) nanoparticles.

It has been found that the protein corona of a particle migrating from one biological fluid to another carries the “fingerprint” of the prior environment.5 This suggests that the route of nanoparticle entry, e.g., inhalation, intravenous injection, or oral ingestion, can influence the composition of the protein corona downstream. Furthermore, the flow rates vary from capillaries to arteries, giving rise to sheer stress and catch and slip bonds associated with the margination, endothelial interaction and extravasation of the nanoparticles within the blood vessels. Indeed, it has been shown that in vivo and in vitro protein coronas differ in both protein type and abundance.6 However, owing to the technical challenge of extracting the protein coronas, it is unknown how these entities develop and evolve in vivo. Furthermore, the residence times of the soft coronas may alter drastically according to the specific microenvironment, including flow. In that regard, establishing 3D-printed tissues and organs as well as virtual model systems may make inroads into unravelling the in vivo protein corona. In an in vivo scenario, a nanoparticle may encounter thousands of different types of proteins. However, because of the finite particle surface area, only hundreds of different types of proteins can bind per nanoparticle at best. Thus, it is expected that individual nanoparticles would possess different protein coronas. Will subpopulations of nanoparticles with different protein coronas behave differently in vivo?

THE PROTEIN CORONA – STEALTH POLYMERS FOR NANOMEDICINE

The future of nanomedicine hinges on its translational value, which at the present stage is mired by the low efficacy of nanoparticle delivery,7 and concerns about potential long-term toxicity. Among the many contributing factors, such as the physical barriers and changing physiological conditions of tissues and organs, the persistence and dynamics of the protein corona may partially account for the compromised targeting of nanomedicines, in particular by stimulating opsonisation.8 To remedy this situation, a major strategy relies on grafting nanoparticles with hydrophilic “stealth” polymers – such as polyethylene glycol (PEG) – against the recognition of opsonins to ensure nanoparticle circulation before reaching its target.9,10 However, PEG is not biodegradable and its repeated dosage and accumulation can give rise to accelerated blood clearance. A recent study revealed that polystyrene nanoparticles grafted with linear PEG or poly(ethyl ethylene phosphate) were surface-enriched with clusterin, which prevented uptake of the nanoparticles by macrophages.11 In addition, biomimetic phosphorylcholine displayed an affinity for apolipoproteins, whereas PEG brushes were associated with complement factors when grafted onto superparamagnetic iron oxide nanoparticles (SPIONs).12 Together, these studies suggest that the stealth capacity of polymers may be convoluted with their nanoparticle substrates. The protein corona also can play a role on the efficacy of imaging contrast agents: for example, the protein corona of fetal bovine serum exerted no effect on the relaxivity of uncharged SPIONs, whereas it modestly increased or significantly decreased the relaxivity of negatively or positively charged SPIONs,13 respectively.

As the endocytic pathway is the major route of nanoparticle cellular uptake, the acidic and enzymatic environments of endosomes and lysosomes likely modulate the protein corona in vitro and in vivo. In this regard, pH- and temperature-responsive “smart” polymers and/or copolymers incorporating stealth moieties as well as multilayered polymeric nanoparticles possessing a range of properties14 may prove advantageous for controlled drug release. The protein corona may also be used to load drugs through self-assembly. Polymers possess specific rigidity that also depends on local pH and ionic strength, whereas interactions between linear or brushed polymers and globular proteins may influence the architecture and hence the physicochemical properties of the protein corona. This provides opportunities for the integration of polymer dynamics with the bio–nano interface for fundamental research. Clearly, developing stealth polymers and novel nanoparticle architectures is an important research domain for improving targeting and drug delivery, for example by exploiting the protein corona by recruiting specific endogenous biomolecules to the nanoparticle surface.15

THE PROTEIN CORONA – THEORETICAL AND COMPUTATIONAL EFFORTS

The theoretical foundation of the protein corona may trace back to the rich literature on protein adsorption, with the use of nanoparticles with high surface curvature replacing planar substrates. Examples of theoretical and computational developments in this area include (1) an adopted Hill model for extracting the equilibrium dissociation and kinetic coefficients for one or two protein species binding with one type of nanoparticle,16 (2) a dynamic model for predicting the evolution and equilibrium composition of the corona based on affinities, stoichiometries and rate constants,17 (3) statistical modelling of quantitative structure–activity relation (QSAR) based on the endpoints of toxicity, blood circulation and biodistribution of nanoparticles, and (4) statistical modelling of biological surface adsorption index (BSAI) based on a multivariate linear regression algorithm and experimentally obtained binding coefficients for small molecules interacting with nanoparticles.18 In addition, atomistic and coarse-grained molecular dynamics simulations can provide molecular-to-particle-level details of nanoparticle–protein interactions that are otherwise difficult to observe experimentally or unavailable from dynamic and statistical modelling. However, as molecular dynamic simulations are typically limited to nanometer-sized systems and the nanosecond timescale, whereas the protein corona is formed in vitro over minutes to days, combining experimental approaches with statistical modelling and multiscale simulations is necessary for extracting the characteristics of both single and ensemble protein coronas. With the rapid improvement of computational power, the coming decade should provide advances in understanding of the bio–nano interface resulting from mathematical modelling and computer simulations. This interdisciplinary fusion is especially welcome, considering that much has already been learned from in silico studies of protein folding and misfolding.

THE PROTEIN CORONA, EXTRAPOLATED

The generic description of the protein corona may require modifications for describing the adsorption of a host of biomolecular species (e.g., proteins, lipids, nucleic acids, sugars, and small molecules) onto nanoparticles. In the case of polymeric nanoparticles, for example, proteins may partition into the polymer interiors through hydrophobic interactions and hydrogen bonding. Another field that may overlap with research on the protein corona is amyloidogenesis, where nanoparticles have shown potency in inhibiting the toxicity and amyloid aggregation of proteins to prevent cell degeneration.19 Interestingly, human islet amyloid polypeptide (IAPP, associated with type 2 diabetes) originating in the pancreas has been detected in the brain and can promote the aggregation of amyloid beta, a hallmark of Alzheimer’s disease. It may be possible that during transport in the body, IAPP aggregates into amyloid fibrils and binds to plasma or cellular proteins to assume new biological and pathological identities.

Perhaps unintended by the authors of the 2007 PNAS paper,1 the term of protein corona has been extrapolated from biological fluids to the vast ecosphere, now under a broader term of “biocorona”. This term has already inspired research on the fate of nanoparticles discharged into soils, plants, surface and groundwater or propagating through tropic transfer.20 Indeed, natural organic matter and aquatic exudates are amphiphiles prevalent in the environment, which can readily modify nanoparticles through mechanisms established for the protein corona. Unsurprisingly, the formation of the biocorona has been found to alter the mobility and toxicity of nanoparticles, analogous to the transformation of nanoparticles in a biological milieu.

SUMMARY AND OUTLOOK

To a large extent, the intricacy of the protein corona stems from the complexity of the biological system, resulting from the necessity of nanoparticles to minimize their surface energy. Understanding, mitigating, and harnessing the biological–synthetic duality of the protein corona are logical steps required for fulfilling many of the biomedical applications intended for nanoparticles. Paradoxically, coating nanoparticle surfaces with hydrophilic polymers can prevent some proteins from adsorbing/fouling, but this can also evoke recognition of the nanoparticles by the immune system, especially with repeated dosing. Alternatively, precoating or aging nanoparticle surfaces with proteins (e.g., clusterin or serum albumin) could confer stealth properties to the nanoparticles, but may subsequently diminish the targeting capacity of the nanoparticles. Yet, opportunities exist to find a compromise through the engineering of, for example, Janus nanoparticles to accommodate stealth polymers with targeting moieties as well as proteins, before introducing the treated nanoparticles to a given biological system. Alternatively, nanoparticles may be rendered “stealthy” by pre-exposure to natural amphiphiles of food sources, such as whey proteins (e.g., caseins, with chaperone-like capacity against protein misfolding, or beta-lactoglobulin) and fat (e.g., lecithin), to fend off the protein corona and evade blood elimination. Furthermore, dosing biological systems with nanoparticles possessing the same core but different surface coatings may be a way to frustrate interactions with immune cells, which could prolong their circulation. With much knowledge gained on the protein corona over the past decade – thanks to multidisciplinary efforts from materials science, chemistry, engineering, biology, immunology, toxicology, physics, and nanomedicine – coupled with increasingly advanced engineering and the predictive, screening and virtual reality power of computational tools, the coming decade is anticipated to bring exciting breakthroughs in theranostics, treatment and prevention of diseases with precise, smart, and environmentally responsible nanotechnologies.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: fcaruso@unimelb.edu.au (F.C.).

*E-mail: thomas.p.davis@monash.edu (T.P.D.).

Notes

The authors declare no competing financial interest

ACKNOWLEDGMENTS

This work was supported by the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology (Project No. CE140100036) and by the Deutsche Forschungsgemeinschaft (DFG, DFG Grant PA 794/25-1). FC and TPD acknowledge Australian Laureate Fellowships from the Australian Research Council.

REFERENCES

1. 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.

2. Docter, D.; Westmeier, D.; Markiewicz, M.; Stolte, S.; Knauer, S. K.; Stauber, R. H. The Nanoparticle Biomolecule Corona: Lessons Learned – Challenge Accepted? Chem. Soc. Rev. 2015, 44, 6094–6121.

3. Monopoli, M. P.; Aberg, C.; Salvati, A.; Dawson, K. A. Biomolecular Coronas Provide the Biological Identity of Nanosized Materials. Nat. Nanotechnol. 2012, 7, 779–786.

4. Vroman, L. Effect of Adsorbed Proteins on the Wettability of Hydrophilic and Hydrophobic Solids. Nature 1962, 196, 476–477.

5. Lundqvist, M.; Stigler, J.; Cedervall, T.; Berggåd, 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.

6. Hadjidemetriou, M.; Al-Ahmady, Z.; Mazza, M.; Collins, R. F.; Dawson, K.; Kostarelos, K. In Vivo Biomolecule Corona around Blood-Circulating, Clinically Used and Antibody-Targeted Lipid Bilayer Nanoscale Vesicles. ACS Nano 2015, 9, 8142–8156.

7. Wilhelm, S.; Tavares, A. J.; Dai, Q.; Ohta, S.; Audet, J.; Dvorak, H. F.; Chan, W. C. W. Analysis of Nanoparticle Delivery to Tumours. Nat. Rev. Mater. 2016, 1, 1–12.

8. Chen, F.; Wang, G.; Griffin, J. I.; Brenneman, B.; Banda, N. K.; Michael Holers, V.; Backos, D. S.; Wu, L. P.; Moein Moghimi, S.; Simberg, D. Complement Proteins Bind to Nanoparticle Protein Corona and Undergo Dynamic Exchange In Vivo. Nat. Nanotechnol. 2017, 12, 387–393.

9. Yan, Y.; Gause, K. T.; Kamphuis, M. M. J.; Ang, C.-S.; O’Brien-Simpson, N. M.; Lenzo, J. C.; Reynolds, E. C.; Nice, E. C.; Caruso, F. Differential Roles of the Protein Corona in the Cellular Uptake of Nanoporous Polymer Particles by Monocyte and Macrophage Cell Lines. ACS Nano 2013, 7, 10960–10970.

10. Pelaz, B.; del Pino, P.; Maffre, P.; Hartmann, R.; Gallego, M.; Rivera-Fernández, S.; de la Fuente, J. M.; Ulrich Nienhaus, G.; Parak, W. J. Surface Functionalization of Nanoparticles with Polyethylene Glycol: Effects on Protein Adsorption and Cellular Uptake. ACS Nano 2015, 9, 6996–7008.

11. Schöttler, S.; Becker, G.; Winzen, S.; Steinbach, T.; Mohr, K.; Landfester, K.; Mailänder, V.; Wurm, F. R. Protein Adsorption Is Required for Stealth Effect of Poly(Ethylene Glycol)- and Poly(Phosphoester)-Coated Nanocarriers. Nat. Nanotechnol. 2016, 11, 372–377.

12. Wang, M.; Siddiqui, G.; Gustafsson, O. J. R.; Käkinen, A.; Javed, I.; Voelcker, N. H.; Creek, D. J.; Ke, P. C.; Davis, T. P. Plasma Proteome Association and Catalytic Activity of Stealth Polymer-Grafted Iron Oxide Nanoparticles. Small 2017, 13, 1701528.

13. Amiri, H.; Bordonali, L.; Lascialfari, A.; Wan, S.; Monopoli, M. P.; Lynch, I.; Laurentg, S.; Mahmoudi, M. Protein Corona Affects the Relaxivity and MRI Contrast Efficiency of Magnetic Nanoparticles. Nanoscale 2013, 5, 8656–8665.

14. Cui, J.; Richardson, J. L.; Bjornmalm, M.; Faria, M.; Caruso, F. Nanoengineered Templated Polymer Particles: Navigating the Biological Realm. Acc. Chem. Res. 2016, 49, 1139–1148.

15. Dai, Q.; Bertleff-Zieschang, N.; Braunger, J. A.; Björnmalm, M.; Cortez-Jugo, C.; Caruso, F. Particle Targeting in Complex Biological Media. Adv. Healthcare Mater. 2017, 1700575.

16. del Pino, P.; Pelaz, B.; Zhang, Q.; Maffre, P.; Ulrich Nienhaus, G.; Parak, W. J.; Protein Corona Formation around Nanoparticles – From the Past to the Future. Mater. Horiz. 2014, 1, 301–313.

17. Dell’Orco, D.; Lundqvist, M.; Oslakovic, C.; Cedervall, T.; Linse, S. Modeling the Time Evolution of the Nanoparticle-Protein Corona in a Body Fluid. PLoS One 2010, 5, e10949.

18. Xia, X. R.; Monteiro-Riviere, N. A.; Riviere, J. E. An Index for Characterization of Nanomaterials in Biological Systems. Nat. Nanotechnol. 2010, 5, 671–675.

19. Ke, P. C.; Sani, M.-C.; Ding, F.; Kakinen, A.; Javed, I.; Separovic, F.; Davis, T. P.; Mezzenga, R. Implications of Peptide Assemblies in Amyloid Diseases. Chem. Soc. Rev. 2017, 46, 6492–6531.

20. Lin, S.; Mortimer, M.; Chen, R.; Kakinen, A.; Riviere, J. E.; Davis, T. P.; Ding, F.; Ke, P. C. NanoEHS beyond Toxicity – Focusing on Biocorona. Environ. Sci.: Nano 2017, 4, 1433–1454.

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