In Situ Characterization of Protein Corona Formation on Silica Microparticles Using Confocal Laser Scanning Microscopy Combined with Microfluidics

Abstract

In biological fluids, proteins bind to particles, forming so-called protein coronas. Such adsorbed protein layers significantly influence the biological interactions of particles, both in vitro and in vivo. The adsorbed protein layer is generally described as a two-component system comprising "hard" and "soft" protein coronas. However, a comprehensive picture regarding protein corona structure is lacking. Herein, we introduce an experimental approach that allows for in situ monitoring of protein adsorption onto silica microparticles. The technique, which mimics flow in vascularized tumors, combines confocal laser scanning microscopy with microfluidics and allows the study of the time-evolution of protein corona formation. Our results show that protein corona formation is kinetically divided into three different phases: phase 1, proteins irreversibly and directly bound (under physiologically relevant conditions) to the particle surface; phase 2, irreversibly bound proteins interacting with pre-adsorbed proteins, and phase 3, reversibly bound "soft" protein corona proteins. Additionally, we investigate particle-protein interactions on lowfouling zwitterionic-coated particles where the adsorption of irreversibly bound proteins does not occur, and on such particles only a "soft" protein corona is formed. The reported approach offers the potential to define new state-of-the art procedures for kinetics and protein fouling experiments. 9 Depending on the characterization method used, the protein corona is described according to either the Gibbs free energy ΔG, 8,10-12 which defines the adsorption and desorption rates of proteins, or binding force 13,14 between the proteins and particle surface. Proteins with a large ΔG have a low probability of desorption and therefore remain associated with the particle surface. These proteins are considered to form the "hard" protein corona. Distinction based on binding forces implies that "hard" protein corona proteins interact directly with the particle surface through long-range, strong protein-surface interactions, whereas proteins in the "soft" protein corona interact with other proteins through short-range, weak protein-protein interactions. Another theoretical distinction is based on the persistence of the protein to remain adsorbed throughout the nanoparticle's journey (i.e. from bloodstream to tissue and past-endocytic environments) as protein corona composition changes during biophysical events. 6-8,15,16 The concept of "persistent" proteins 5 originates from studies where the "hard" protein corona is used to follow the particle's past. 17-19 It is becoming increasingly important to clearly understand the complex process of protein corona formation, with a focus on the influence of the "soft" protein corona on physiological interactions. 4,7,13,20-24 However, to do so, it is crucial to acquire and understand further details such as the time-evolution of protein corona formation. Existing techniques for investigating the protein corona can be divided into ex situ and in situ ...

Glycogen as a Building Block for Advanced Biological Materials

“…Most pertinently, studies on the in vivo fate of glycogen particles of different size, molecular weight, and surface functionalization, particularly with regards to immune system stimulation, are needed. Of emerging importance is the role of the protein corona on determining the in vivo fate of glycogen nanoparticles, and how this may, or may not, contribute to immune system recognition, and in vivo biodistribution. In this context, we are investigating the use of hydrophilic peptides to improve the pharmacokinetics and biodistribution of functionalized glycogen nanoparticles.…”

“…Furthermore, glycogen particles in vivo are associated with a population of proteins that mediate their biological function . Given that the “hard” protein corona can only be removed from particles under extreme alkaline conditions, it stands to reason that there remain associated proteins on commercially available glycogen, particularly embedded proteins such as glycogenin. This can give rise to fluorescence of the nanoparticles and may provide an anchor point to further modify the nanoparticles through polymerization …”

Mapping and identification of soft corona proteins at nanoparticles and their impact on cellular association

“…At the surface of nanoparticles, proteins can undergo reorientation and conformational changes 17 , 18 , presumably leading to at least a partially denatured state that has a reduced dissociation rate in a process referred to as “hardening” 19 . The evolution and dynamics of HC formation are relatively well studied 14 , 20 – 22 ; HC is established rapidly, and the evolution of HC over time is only quantitative with altered relative amounts, rather than the changes in protein composition expected from the Vroman effect 12 . The current understanding is that the HC proteins—with their long residence time—give the nanoparticles a biological identity by presenting receptor-binding sites for cellular interactions with a biologically relevant timescale 23 .…”

Particle-by-Particle in Situ Characterization of the Protein Corona via Real-Time 3D Single Particle Tracking Microscopy

“…Due to the difficulties in preserving the highly dynamic "soft" corona upon extracting NPs from the biological fluid, very few studies have been done to directly monitor these interactions. [29,31,40] To observe this soft corona using fluorescence, all proteins in the solution need to be fluorescently labeled, creating an enormous background signal, particularly at biologically relevant protein concentrations, burying the far smaller corona signal in the noise (Figure 4a, c). Some previous imaging studies employed a partial labeling strategy (~1%) that may generate significant soft corona quantification error and is limited to low and physiologically irrelevant protein concentrations.…”

“…To overcome the intrinsic uncertainty in diffusion-based in situ methods, recent studies have utilized imaging-based strategies to directly observe single NP-protein interactions with high sensitivity. [8,[28][29][30][31] While far more sensitive than extracting a diffusion coefficient, each of these methods required immobilization of the NP for sufficient characterization. The change of dynamics of the NP and the interaction between NP and surface may strongly affect protein binding, and it is difficult, if not impossible, to distinguish NP-bound proteins from surface-bound proteins.…”