Inside the Protein Corona: From Binding Parameters to Unstained Hard and Soft Coronas Visualization

Abstract

Proteins spontaneously adsorb on nanoparticle surfaces when injected into the bloodstream. It drastically modifies the nanoparticle's fate and how they interact with organs and cells. Although this protein layer (protein corona) has been widely studied, the robustness of the most employed characterization methods and the visualization of its unstained fractions remain open questions. Here, synchrotron-based small-angle X-ray scattering was used to follow the corona formation and estimate binding parameters. At the same time, transmission electron microscopy under cryogenic conditions associated with crosscorrelation image processing and energy-filtered transmission electron microscopy allowed to determine protein corona morphology and thickness together with the visualization of its unstained hard and soft fractions. The above-presented strategy shows tremendous potential for deciphering fundamental protein corona aspects and can contribute to rational medical nanoparticle engineering.

Probing the protein corona of gold/silica nanoparticles by Taylor dispersion analysis-ICP-MS

“…The formation of a BSA corona on silica NPs was studied by other groups and seems to be size-dependent. A corona thickness of 7 nm and 4 nm was reported by Galdino et al for 40 nm and 80 nm -silica NPs respectively [17]. Monopoli et al observed a corona thickness of 4 nm for 200 nm silica NPs [27].…”

“…Based on microscopy data, this was attributed by Galdino et al to the formation of small aggregates [17]. As TDA experiments showed that FET aggregation did not occur in phosphate buffer using such a concentration (see Supplementary material, section 2), NPs aggregation mediated by proteins might be considered, even if the formation of protein multilayers cannot be excluded in the absence of additional proofs.…”

“…They successfully measured corona thicknesses of 4 -9 nm. Small-angle X-ray scattering (SAXS) was also used to determine binding parameters in the presence of both BSA and serum [17]. Although very sensitive, SAXS revealed insufficiently accurate measurements of the protein corona thickness.…”

“…et al described both reversible and irreversible adsorption of BSA onto silica NPs upon incubation in a BSA solution at 60 µM (4.16 mg.mL -1 ). A thin layer of protein corona (~ 4nm) was still observed by energy-filtered transmission electron microscopy (EF-TEM) around the silica NPs after several washing steps[17]. Norde et al also reported an irreversible adsorption of BSA on silica upon dilution after a 20h-incubation[33].…”

Synchrotron radiation-based analysis of interactions at the nano–bio interface

“…This strategy validates the applicability of TEM methodologies to decipher fundamental PC aspects and its behavior in simple and complex media, which can contribute to rational design of nanomedicines. 69 Ptychographic X-ray computed tomography (PXCT) is an extension of X-ray imaging technique to perform non-invasive and lens-free imaging of protein structures at the membrane interface with quantitative electron density contrast at sharp resolutions of 15 nm, 70 which can be used to interpret PC and nano–bio interactions at the nanoscale level. Biological samples (tissue, organelle) are processed and prepared for cryo X-ray imaging (with a potential penetration of 100 nm thickness across the sample).…”