Opinion
The protein corona on nanoparticles as viewed from a nanoparticle-sizing perspective Haixia Wang,1 Youhui Lin,1,2 Karin Nienhaus1 and G. Ulrich Nienhaus1,2,3,4* Most surfaces of engineered nanoparticles (NPs) are reactive toward biomolecules. Therefore, whenever NPs become immersed in biological fluids, proteins and other biomolecules bind to the NP surface, forming an adsorption layer (biomolecular corona) that modifies the NPs’ physicochemical properties and subsequent interactions with living systems. Its exploration is a formidable endeavor owing to the enormous diversity of engineered NPs in terms of their physicochemical properties and the vast number of biomolecules available in biofluids that may bind to NPs with widely different strengths. Significant progress has been made in our understanding of the biomolecular corona, but even very basic issues are still controversially debated. In fact, there are divergent views of its microscopic structure and dynamics, even on physical properties, such as its thickness. As an example, there is no agreement on whether proteins form monolayers or multilayers upon adsorption. In our quantitative studies of NP– protein interactions by in situ fluorescence correlation spectroscopy (FCS) with highly defined model NPs and important serum proteins, we have universally observed protein monolayer formation around NPs under saturation or even oversaturation conditions. Here, we critically discuss biomolecular corona characterization using FCS and dynamic light scattering and identify challenges and future opportunities. Further careful, quantitative experiments are needed to elucidate the mechanisms of biomolecular corona formation and to characterize its structure. © 2017 Wiley Periodicals, Inc. How to cite this article:
WIREs Nanomed Nanobiotechnol 2017, e1500. doi: 10.1002/wnan.1500
INTRODUCTION
R
apid advances in nanoscience and nanotechnology have led to diverse and widespread applications of engineered nanoparticles (NPs). A detailed
*Correspondence to:
[email protected] 1
Institute of Applied Physics, Karlsruhe Institute of Technology, Karlsruhe, Germany
2
Institute of Nanotechnology, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany
3
Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Eggenstein-Leopoldshafen, Germany
4
Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA Conflict of interest: The authors have declared no conflict of interests for this article.
understanding of NP interactions with cells, tissues, and organisms is indispensable for their safe application as diagnostic and therapeutic devices in nanomedicine and also to minimize hazards due to their unintended, yet unavoidable release into the environment.1,2 In the complex biological environment of a living organism, proteins and other biomolecules in biological fluids (e.g., blood and lung-lining fluid) spontaneously form an adsorption layer on the NP surface, the so-called biomolecular corona.3–7 Its properties need to be known in detail because it is this adsorption layer rather than the NP surface that mediates interactions with the living organism and governs physiological responses, including cellular internalization, biodistribution, and toxicity.8 Despite enormous efforts devoted to the characterization of
© 2017 Wiley Periodicals, Inc.
1 of 9
Opinion
wires.wiley.com/nanomed
the corona in recent years, there is still considerable debate about key physicochemical properties, including composition, thickness, and temporal evolution of the adsorption layer. Characterization of the biomolecular corona is a formidable endeavor due to the complexity of biofluids as well as the enormous diversity of NPs that are currently being synthesized. Biofluids contain a vast number of different biomolecules, especially proteins. Globular proteins are colloidally very stable; in fact, a general aggregation tendency of blood proteins would be disastrous for the organism. However, proteins may associate with the non-native surface of a given type of NP with widely different strengths. It is important to note that proteins are weakly stabilized, delicate polypeptide architectures, performing large-scale fluctuations among a vast number of conformations at ambient temperature.9 Thus, interactions between the NP surface and the adsorbing protein may induce major structural changes in the protein, leading to partial or even complete denaturation. Upon unfolding, polypeptide chains of globular proteins expose hydrophobic moieties and thus interact with the NP surface and other proteins in the biofluid in distinctly different ways than native proteins. Moreover, some proteins of the immune system (e.g., opsonins) are intrinsically reactive toward both NP surfaces and other proteins. There is an impressive diversity in the design of engineered NPs in terms of their physical and chemical properties. Some NP surfaces interact only weakly and are thus benign toward proteins, whereas others are highly reactive and cause adsorbing proteins to unfold. Many investigations have shown in detail how NP properties, including size, shape, composition, surface functionalization, and surface charge, can affect biomolecular adsorption.1,3,4,7,10 Many NP surfaces are chemically stable, but a further degree of complexity arises if they change their properties over time, e.g., because of protein binding. For example, weakly bound stabilizing ligands, such as citrate on gold NPs, may be replaced by adsorbing proteins on relevant timescales. Then, the observed processes are not properly described as an evolution of the biomolecular corona but rather a coevolution of the corona with the NP surface. Furthermore, we should also be aware that surface inhomogeneity exists within the NP ensemble and even for an individual NP. In fact, as-prepared NPs may not always resemble the colorful schematic depictions shown in publications. Analytical tools are evidently limited and do not give a complete picture of the physicochemical nature of NP surfaces; so all we can do is to perform exceedingly careful characterizations using 2 of 9
complementary techniques. Because of these caveats, great caution must be exercised when extrapolating from the findings of one study to another and, even more importantly, when venturing to draw general conclusions from specific observations. A vast number of experiments have been carried out in recent years to characterize the biomolecular corona with a wide range of experimental techniques.4,5,11 Whereas different methods often provide complementary information, which may greatly help to obtain a more complete picture, they can also lead to conflicting results if researchers are not familiar with the intricacies of the techniques. For more than a decade, we have been studying protein adsorption onto NPs using fluorescence correlation spectroscopy (FCS), a technique closely related to the widely used dynamic light scattering (DLS). Both techniques yield the diffusion coefficient, which can be recast into the hydrodynamic radius via the Stokes–Einstein relation. Thus, FCS and DLS can, in principle, provide direct access to the linear dimensions of the NPs as they diffuse in the solution. Here, we briefly but critically sketch current views of the protein adsorption layer and discuss these in the context of results from NP-sizing experiments.
CURRENT VIEWS OF PROTEIN ADSORPTION ONTO NANOPARTICLES In the simplest model, protein binding to NPs is described by an equilibrium process governed by on and off rate coefficients (Figure 1). In a complex biofluid such as blood, which contains thousands of different proteins, abundant proteins are expected to associate quickly with the NP but will subsequently Protein-mediated NP aggregation
kon koff Denaturation Protein monolayer
Protein multilayer
F I G U R E 1 | Nanoparticle (NP)–biomolecule interactions. In a biological fluid, proteins may be considered as a cloud around the NP. Some will adsorb reversibly to the NP surface and form a monolayer. Adsorption may be accompanied by (partial) unfolding, which could expose hydrophobic moieties, onto which further proteins may possibly adsorb as a second layer. NPs covered with unfolded proteins may also undergo protein-mediated aggregation. Box: reversible protein binding.
© 2017 Wiley Periodicals, Inc.
WIREs Nanomedicine and Nanobiotechnology
Protein corona on nanoparticles
be replaced by rare ones that bind more tightly, a scenario known as the Vroman effect. Thus, given that the rate coefficients governing protein binding and unbinding are of suitable magnitude, one may observe an initial, short-lived ‘soft corona’ that subsequently develops into a long-lived ‘hard corona.’ In this context, ‘long-lived’ refers to a residence time of the proteins on the NP surface longer than the duration of the experiment (oftentimes hours to days). A large part of experimental protein corona investigations aim at the identification of the types and amounts of proteins forming the corona using a multistep procedure to separate NPs and biofluid: (1) incubation of NPs with a biofluid (e.g., blood serum), (2) separation of unbound proteins from the NPs (e.g., by centrifugation and washing), and (3) removal and (4) identification of the corona proteins (e.g., by chromatography, gel electrophoresis, and mass spectrometry). Clearly, only very tightly binding polypeptides, i.e., those with small off rates, will remain on the NPs during the separation step, whereas the weakly binding ones will be lost. Strong binders are likely not intact proteins, but rather (at least partially) unfolded polypeptide chains.12 Upon denaturation, exposed hydrophobic moieties of the protein may give rise to a tightly adhering polypeptide layer on the NP surface (Figure 1). These deposits can be even further stabilized by covalent interactions to the surface and to other peptide chains (e.g., cysteine thiols binding to metal atoms and reactions involved in opsonization processes). Such strong interactions may completely suppress protein desorption; the corona thus remains static on all relevant timescales and is not appropriately described by protein-binding equilibria. The display of hydrophobic moieties from denatured proteins on the NP surface can give rise to further consequences: (1) colloidal instability causing NP aggregation and (2) enhanced interactions with normally colloidally very stable globular proteins, causing further protein accretion (Figure 1). Importantly, formation of such hard coronas is not captured by an equilibrium model describing protein binding and unbinding; rather, the additional unfolding transition changes the nature of the polypeptide and causes the chain to tightly stick to the surface so that it can no longer desorb. It has been argued that only strongly adhering polypeptides are important for triggering the ensuing cellular responses and that weak binders may not be relevant.3 We do not share this view, considering that cellular uptake has been observed to be markedly different even for NPs that bind proteins only weakly.13,14
In situ measurements of NPs within a biofluid can provide the full picture of NP–protein interactions because weakly adsorbing protein layers will not be lost during the experiment. There are currently only a few methods available that provide in situ data, including FCS and DLS. Even though they yield only a single parameter, i.e., the hydrodynamic radius (RH) of the particle under study, valuable insights into the structure and dynamics of protein adsorption layers can be revealed from precise NP sizing, as we will show below. Depending on the nature of the samples, one may, however, encounter serious pitfalls in the interpretation of the data. With an illustrative case study, we will discuss potential misinterpretations of apparently clear experimental data.
IN SITU QUANTIFICATION OF THE CORONA THICKNESS Fluorescence Correlation Spectroscopy Our group has established FCS as a powerful in situ technique to quantitatively examine NP–protein interactions while NPs are immersed in biological fluids. FCS is based on the analysis of the duration of brief bursts of photons emitted by individual lightemitting particles diffusing through an observation volume of ~1 fL, defined by a focused laser beam and confocal detection in a fluorescence microscope (Figure 2(a)).15–17 Very low concentrations of fluorescent NPs (
