Quantitative profiling of the protein coronas that form around nanoparticles

Abstract

Nanoparticle applications in biotechnology and biomedicine are steadily increasing. In biological fluids, proteins bind to nanoparticles that form the protein corona, crucially affecting the nanoparticles' biological identity. As the corona affects in vitro and/or in vivo nanoparticle applications, we developed a method to obtain time-resolved protein corona profiles formed on various nanoparticles. After incubation in plasma or a similar biofluid, or after injection into a mouse, the first analytical step is sedimentation of the nanoparticle-protein complexes through a sucrose cushion, thereby allowing analysis of early corona formation time points. Next, corona profiles are visualized by gel electrophoresis and quantitatively analyzed after tryptic digestion using label-free liquid chromatography-high-resolution mass spectrometry. In contrast to other approaches, our established methodology allows the researcher to obtain qualitative and quantitative high-resolution corona signatures. The protocol can be readily extended to the investigation of protein coronas from various nanomaterials (as an example, we applied this protocol to different silica nanoparticles (SiNPs) and polystyrene nanoparticles (PSNPs)). Depending on the number of samples, the protocol from nanoparticle-protein complex recovery to data evaluation takes ~8-12 d to complete.

The bio-corona and its impact on nanomaterial toxicity

“…Thus, a protein, which was no 'longer detectable' at a certain time point, was classified as being 'absent' or 'disappearing' in previous studies, thereby contributing to the model of a highly dynamic protein corona (44,61,70,72). Hence, we again want to emphasize that it is highly important to use the highest technological standards and standardized experimental procedures (SOPs) for the determination of protein corona binding kinetics (82), allowing inter-laboratory comparison and model building by further systematic studies. The same study also demonstrated that the plasma protein corona is highly complex, containing over 200 different proteins, and surprisingly established in less than one minute (36).…”

Visualization of the protein corona: towards a biomolecular understanding of nanoparticle-cell-interactions

“…7 This means that the individual proteins present in each case are responsible for regulating the cellular uptake and the intracellular fate. 8,9 The proteomic composition, 10 size and aggregation 11 effects of the protein corona are well-known, but its morphology has still to be examined. The protein corona is usually shown either as a uniform layer or as multiple layers covering the nanoparticle.…”

Technical tip: high-resolution isolation of nanoparticle–protein corona complexes from physiological fluids

“…extended exposure time with the biological fluid during the pellet procedure in the centrifuge (see the ESI † for LC-MS data). 35,56,57 In contrast, the extensive aggregation combined with the complex composition of the intestinal media made it impossible to extract the PC complexes from this environment by conventional methods. Mainly composed of enzymes (trypsin, 23 kDa, and chymotrypsin, 25 kDa) and some persistent peptides, the intestinal environment led to strong NP aggregation but the PC complexes could be isolated through UC.…”

“…9a) presented some notable bands at 21 kDa persistent from the gastric phase and chymotrypsin at 25 kDa. Bile salts caused desorption of proteins according to their concentration and exposure time, 35,58,59 therefore also in this case the ability of UC to limit the contact time between the PC complexes and biological medium may affect the corona composition. Some bands at a higher molecular weight could not be found anywhere else and showed quite regular spacing among them.…”

“…31 Density gradient UC has been extended to purify NPs from the excess of coating agents, 32 and obtain narrow size distributions. 33,34 Recently, Docter, Tenzer and co-workers 35,36 and…”