Concepts and Approaches to Reduce or Avoid Protein Corona Formation on Nanoparticles: Challenges and Opportunities

Abstract

This review describes the formation of a protein corona (or its absence) on different classes of nanoparticles, its basic principles, and its consequences for nanomedicine. For this purpose, it describes general concepts to control (guide/minimize) the interaction between artificial nanoparticles and plasma proteins to reduce protein corona formation. Thereafter, methods for the qualitative or quantitative determination of protein corona formation are presented, as well as the properties of nanoparticle surfaces, which are relevant for protein corona prevention (or formation). Thereby especially the role of grafting density of hydrophilic polymers on the surface of the nanoparticle is discussed to prevent the formation of a protein corona. In this context also the potential of detergents (surfactants) for a temporary modification as well as grafting-to and grafting-from approaches for a permanent modification of the surface are discussed. The review concludes by highlighting several promising avenues. This includes (i) the use of nanoparticles without protein corona for active targeting, (ii) the use of synthetic nanoparticles without protein corona formation to address the immune system, (iii) the recollection of nanoparticles with a defined protein corona after in vivo application to sample the blood proteome and (iv) further concepts to reduce protein corona formation.

Keywords: nano particles; nanomedicine; protein corona; surface modification.

Figure 1

(left) Sketch of various nanoparticles (colloids, polymeric micelles, liposomes, and nanogels), which are used in the context of nanomedicines. They differ in the accessibility of their inner core (mostly hydrophobic, reddish) to proteins as outlined in ref. [86]. In typical colloids (inorganic or organic) the inner, solid core may be coated with detergents (blue). But as the solubility of the detergents in water is high, they can ‐to a certain extent‐ diffuse away from the colloidal surface enabling protein adsorption. Polymeric micelles are, however, characterized with a more stable and denser hydrophilic polymer corona than colloidal nanoparticles or liposomes. It hinders/prevents the access of plasma proteins to the hydrophobic core by entropic shielding. Liposomes, as models of cellular structures, are also not as well protected by entropic shielding (only a few long hydrophilic chains from PEG). However, their surface is covered covalently with highly hydrophilic head groups (mostly zwitter‐ionic) with pronounced hydration shell, which can reduce protein corona formation. Nanogels differ from the other systems, because they are strongly hydrated throughout the whole nanostructure and thus, they miss any interface to a hydrophobic inner core, which could act as a nucleus for protein corona formation. (right) Schematic sketch of the formation of a protein corona on purely protected nanoparticles (e.g., colloids). On the other side the hydrophilic polymer corona in core crosslinked micelles prevents the approaching of nanoparticles to the hydrophobic core.

Figure 2

Formation of a protein corona and its prevention on surfaces; Please consider that plasma proteins tend to segregate into an inner hydrophobic core and a more hydrophilic surface (left side). So, they have an amphiphilic, colloidal character by themselves. Thus, they tend to form protein layers (often in combination with a change of their conformation) on solid surfaces, which are poorly coated (A). However, particles with a complete surface coverage (B) may avoid protein corona formation.

Figure 3

Self‐assembly of amphiphilic block copolymers into polymer micelles (compare Figure 1) ‐as well as‐ their interaction with plasma proteins. If the inner core is shielded by a dense corona of hydrophilic and protein‐resistant polymers, attractive interaction sites, e.g., the hydrophobic core, are not accessable for plasma proteins. For micelles, however, the equilibrium between micelles (middle) and unimers (left) is of major importance. It depends on the critical micelle concentration. Individual unimers are of amphiphilic nature and therefore can interact directly with plasma proteins in solution (see left, I). Alternatively an exchange reaction between unimers and proteins can occur leading to the integration of proteins into polymer micelles (see right, II).

Figure 4

Workflow to identify type and amount of nanoparticles associated proteins. It starts with a separation step, which separates aggregates, nanoparticles, and proteins according to size (or mass) and allows to isolate the fractions (like AF4, upper line). Analysis can then be done by combining dynamic light scattering (size) and electrophoresis (separation and quantification of the proteins). At last LC‐MS allows the identification of the proteins and their relative amounts to distinguish really accumulated proteins from proteins, which are just “co‐eluting”. Reproduced with permission.^{[ 71 ]} Copyright 2020, Wiley‐VCH GmbH.

Figure 5

Results of the separation of nanoparticles from plasma proteins (core crosslinked polymer micelles and polymer brushes from ref. [71]) according to size by AF4 (A) (see Figure 4 for the complete workflow) and detection of proteins in the different fractions (B: Coomassie staining and C: silver staining).