In biological fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living systems. As physiological systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biological relevancy. Here we show that label-free snapshot proteomics can be used to obtain quantitative time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to form rapidly (<0.5 minutes) and, over time, to change significantly in terms of the amount of bound protein, but not in composition. Rapid corona formation is found to affect haemolysis, thrombocyte activation, nanoparticle uptake and endothelial cell death at an early exposure time.
In biological fluids, proteins associate with nanoparticles, leading to a protein "corona" defining the biological identity of the particle. However, a comprehensive knowledge of particle-guided protein fingerprints and their dependence on nanomaterial properties is incomplete. We studied the long-lived ("hard") blood plasma derived corona on monodispersed amorphous silica nanoparticles differing in size (20, 30, and 100 nm). Employing label-free liquid chromatography mass spectrometry, one- and two-dimensional gel electrophoresis, and immunoblotting the composition of the protein corona was analyzed not only qualitatively but also quantitatively. Detected proteins were bioinformatically classified according to their physicochemical and biological properties. Binding of the 125 identified proteins did not simply reflect their relative abundance in the plasma but revealed an enrichment of specific lipoproteins as well as proteins involved in coagulation and the complement pathway. In contrast, immunoglobulins and acute phase response proteins displayed a lower affinity for the particles. Protein decoration of the negatively charged particles did not correlate with protein size or charge, demonstrating that electrostatic effects alone are not the major driving force regulating the nanoparticle-protein interaction. Remarkably, even differences in particle size of only 10 nm significantly determined the nanoparticle corona, although no clear correlation with particle surface volume, protein size, or charge was evident. Particle size quantitatively influenced the particle's decoration with 37% of all identified proteins, including (patho)biologically relevant candidates. We demonstrate the complexity of the plasma corona and its still unresolved physicochemical regulation, which need to be considered in nanobioscience in the future.
In biological fluids, proteins may associate with nanoparticles (NPs), leading to the formation of a so-called "protein corona" largely defining the biological identity of the particle. Here, we present a novel approach to assess apparent binding affinities for the adsorption/desorption of proteins to silver NPs based on the impact of the corona formation on the agglomeration kinetics of the colloid. Affinities derived from circular dichroism measurements complement these results, simultaneously elucidating structural changes in the adsorbed protein. Employing human serum albumin as a model, apparent affinities in the nanomolar regime resulted from both approaches. Collectively, our findings now allow discrimination between the formation of protein mono- and multilayers on NP surfaces.
with cells, [10] can shield recognition units, [15] but its role for biodistribution and circulation is still far from well understood. [8,16] Except for liposomes, other types of nano-sized drug delivery systems, such as polymeric micelles or more complex polymer constructs like cylindrical polymer brushes [17] have been so far hardly investigated with respect to protein corona formation (for comparison between the structures of these nanoparticles and the colloidal nanoparticles discussed above, see Figures S1 and S2 in the Supporting Information). [8,[18][19][20] However, the former are interesting, since polymeric micelles are in advanced stages of clinical testing (e.g., CPC634 (phase II) [21] and NC-6004 Nanoplatin (phase III) [22] ).To evaluate the formation of a protein corona, separation of the nanoparticle-protein-complex from unbound proteins used for incubation or upon in vivo exposure becomes a necessity. The isolation of incubated nanoparticles (colloids and inorganic nanoparticles) from unbound proteins was mainly performed by centrifugation, which is a separation method based on differences in density. [23] Thus, this method allows only the purification of nanoparticles with a higher density but can hardly be used for low density particles, such as polymeric micelles and polymer brushes. [24] Only recently, size exclusion techniques such as size exclusion chromatography and asymmetrical flow field-flow fractionation (AF4) have been employed for this purpose. [19,25,26] AF4 is a separation technique, which can be applied for the separation of particle and protein mixtures in the size range between 1 nm and 1 µm. [27,28] It consists of a separation channel with a flow gradient in which the particles are separated by an additional vertical force field depending on their diffusion coefficient. [29,30] During an AF4 measurement, the injected particles are pushed by the vertical cross flow toward the membrane at the bottom of the channel. Due to Brownian motion, the particles are diffusing back into the middle of the channel. Since smaller particles are faster than larger ones (because of their higher diffusion coefficient), they are concentrating faster in the middle, thus eluting first through the channel outlet. In contrast to conventional size exclusion chromatography, in AF4 the contact to the interface and the shear forces are substantially reduced, which leads to very mild separation conditions, minimizing perturbations of a potential protein corona. [26,31] Based on this method, Landfester and coworkers recently fully characterized the protein corona of Lutensol AT50-coated polystyrene nanoparticles and PEG functionalized liposomes, identifying all adsorbed proteins. [19,26] Here, we present a purification procedure based on AF4 for the separation of smaller polymeric architectures (R h : 20-30 nm) that are hardly separable by centrifugation. We isolated polymeric nanoparticles from unbound blood plasma components and characterized them by dynamic light scattering, gel electrophoresis and mass s...
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