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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 origins and time evolution of the protein corona, differences in the nanoparticle−protein entity in 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: “SOFT” VERSUS “HARD”, IN VITRO VERSUS IN VIVO
THE PROTEIN CORONA: A BRIEF HISTORY A decade has passed since the inception of the protein corona, as coined by Dawson, Linse, and co-workers.1 Although the adsorption of proteins onto surfaces, including particles, has long been 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 the biological milieu. However, 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, and 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. © 2017 American Chemical Society
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 Upon protein binding, which is energetically favored, enthalpy is reduced and 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; second, 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; last, but perhaps most importantly, they may mask the chemical or biological functionalities imparted to the nanoparticle in the laboratory (Figure 1). Published: December 5, 2017 11773
DOI: 10.1021/acsnano.7b08008 ACS Nano 2017, 11, 11773−11776
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Cite This: ACS Nano 2017, 11, 11773−11776
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Perspective
may partially account for the compromised targeting of nanomedicines, in particular, by stimulating opsonization.8 To remedy this situation, one major strategy relies on grafting nanoparticles with hydrophilic “stealth” polymerssuch 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 surfaceenriched 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 in 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.
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 nanoparticle (illustrated as aqua blue fibrils).
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.
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 result 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 stresses and catchand-slip bonds associated with the margination, endothelial interaction, and extravasation of the nanoparticles within 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 change dramatically according to the specific microenvironment, including flow. In that regard, establishing three-dimensional (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 most. Thus, it is expected that individual nanoparticles would possess different protein coronas. Will subpopulations of nanoparticles with different protein coronas behave differently in vivo?
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 influence provides opportunities for the integration of polymer dynamics with the bio−nano interface for fundamental research. 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: 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 delivery7 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
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 11774
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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.
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 modeling of quantitative structure−activity relationship (QSAR) based on the end points of toxicity, blood circulation, and biodistribution of nanoparticles; and (4) statistical modeling 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 modeling. However, as molecular dynamic simulations are typically limited to nanometer-sized systems and the nanosecond time scale, whereas the protein corona is formed in vitro over minutes to days, combining experimental approaches with statistical modeling 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 the bio−nano interface resulting from mathematical modeling 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.
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 β-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−largely due to multidisciplinary efforts from materials science, chemistry, engineering, biology, immunology, toxicology, physics, and nanomedicinecoupled 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.
With the rapid improvement of computational power, the coming decade should provide advances in understanding the bio−nano interface resulting from mathematical modeling and computer simulations. 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 “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,
AUTHOR INFORMATION Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Pu Chun Ke: 0000-0003-2134-0859 Sijie Lin: 0000-0002-6970-8221 Wolfgang J. Parak: 0000-0003-1672-6650 Thomas P. Davis: 0000-0003-2581-4986 Frank Caruso: 0000-0002-0197-497X 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 11775
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(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.
Technology (Project No. CE140100036) and by the Deutsche Forschungsgemeinschaft (DFG, DFG Grant PA 794/25-1). F.C. and T.P.D. acknowledge Australian Laureate Fellowships from the Australian Research Council.
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