Complementary analysis of the hard and soft protein corona: sample preparation critically effects corona composition

Abstract

Here we demonstrate how a complementary analysis of nanocapsule-protein interactions with and without application media allows gaining insights into the so called hard and soft protein corona. We have investigated how both human plasma and individual proteins (human serum albumin (HSA), apolipoprotein A-I (ApoA-I)) adsorb and interact with hydroxyethyl starch (HES) nanocapsules possessing different functionalities. To analyse the hard protein corona we used sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and a protein quantitation assay. No significant differences were observed with regards to the hard protein corona. For analysis of the soft protein corona we characterized the nanocapsule-protein interaction with isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). DLS and ITC measurements revealed that a high amount of plasma proteins were adsorbed onto the capsules' surface. Although HSA was not detected in the hard protein corona, ITC measurements indicated the adsorption of an HSA amount similar to plasma with a low binding affinity and reaction heat. In contrast, only small amounts of ApoA-I protein adsorb to the capsules with high binding affinities. Through a comparison of these methods we have identified ApoA-I to be a component of the hard protein corona and HSA as a component of the soft corona. We demonstrate a pronounced difference in the protein corona observed depending on the type of characterization technique applied. As the biological identity of a particle is given by the protein corona it is crucial to use complementary characterization techniques to analyse different aspects of the protein corona.

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

“…31 In addition, it is known that the protein adsorption pattern is highly influenced by surface functionalization of nanoparticles. 24,32,33 We found that the absolute number of bound proteins per defined surface of nanoparticles is significantly higher (Fig. 3C) for negatively charged nanoparticles (PS-COOH) in comparison to un-functionalized (PS), or positively charged nanoparticles (PS-NH 2 ).…”

“…Interestingly, the soft protein corona is not shown by TEM to be as uniformly distributed and wellrounded, but rather as an undefined network surrounding the nanoparticle. Generally 2,20 it is described as highly dynamic layer of proteins which have high exchange rates and low binding affinities towards the nanoparticle. By adding trehalose and uranyl acetate (UA) for TEM sample preparation, the highly dynamic structure is fixed.…”

Protein corona: a new approach for nanomedicine design

“…For example, human serum albumin (HSA) adsorbed on hydroxyethyl starch nanocapsules could be detected by ITC, while there was no trace of HSA when evaluated by SDS-PAGE. 32 Hence, it is pivotal to choose suitable analytical techniques to accurately characterize protein corona properties since protein corona is related with physicochemical properties and biological identity of NPs.…”

Preliminary protein corona formation stabilizes gold nanoparticles and improves deposition efficiency

“…The hard corona is a single layer of proteins directly bound to the NP surface with high affinity, forming a stable coating. The soft corona, which covers the hard corona, is loosely associated and, therefore, promotes dissociation and rapid biomolecule exchange (Winzen et al 2015). Previous studies have demonstrated that the protein corona directly impacts the nano-cellular interface, thereby determining NP deposition efficiency, membrane interactions, degree of endocytosis, and biocompatibility (Chanana et al 2013;Lesniak et al 2013;Treuel et al 2014).…”