Transferrin coated nanoparticles: Study of the bionano interface in human plasma

It is now well established that the surface of nanoparticles (NPs) in a biological environment is immediately modified by the adsorption of biomolecules with the formation of a protein corona and it is also accepted that the protein corona, rather than the original nanoparticle surface, defines a new biological identity. Consequently, a methodology to effectively study the interaction between nanomaterials and the biological corona encountered within an organism is a key objective in nanoscience for understanding the impact of the nanoparticle-protein interactions on the biological response in vitro and in vivo. Here, we outline an integrated methodology to address the different aspects governing the formation and the function of the protein corona of polystyrene nanoparticles coated with Transferrin by different strategies. Protein-NP complexes are studied both in situ (in human plasma, full corona FC) and after washing (hard corona, HC) in terms of structural properties, composition and second-order interactions with protein microarrays. Human protein microarrays are used to effectively study NP-corona/proteins interactions addressing the growing demand to advance investigations of the extrinsic function of corona complexes. Our data highlight the importance of this methodology as an analysis to be used in advance of the application of engineered NPs in biological environments

IgG and fibrinogen driven nanoparticle aggregation

A thorough understanding of how proteins induce nanoparticle (NP) aggregation is crucial when designing in vitro and in vivo assays and interpreting experimental results. This knowledge is also crucial when developing nano-applications and formulation for drug delivery systems. In this study, we found that extraction of immunoglobulin G (IgG) from cow serum results in lower polystyrene NPs aggregation. Moreover, addition of isolated IgG or fibrinogen to fetal cow serum enhanced this aggregation, thus demonstrating that these factors are major drivers of NP aggregation in serum. Counter-intuitively, NP aggregation was inversely dependent on protein concentration; i.e., low protein concentrations induced large aggregates, whereas high protein concentrations induced small aggregates. Protein-induced NP aggregation and aggregate size were monitored by absorbance at 400 nm and dynamic light scattering, respectively. Here, we propose a mechanism behind the protein concentration dependent aggregation; this mechanism involves the effects of multiple protein interactions on the NP surface, surface area limitations, aggregation kinetics, and the influence of other serum proteins.We thank Professor Sara Linse for scientific discussions and advice and Professor Patrik Brundin for enabling access to the light microscope. The project received financial support from Nanometer structure consortium at Lund University (nmC@LU), Lars Hierta Foundation, and the research school FLAK of Lund University

Reversible <i>versus</i> Irreversible Binding of Transferrin to Polystyrene Nanoparticles: Soft and Hard Corona

Protein adsorption to nanoparticles (NPs) is a key prerequisite to understand NP–cell interactions. While the layer thickness of the protein corona has been well characterized in many cases, the absolute number of bound proteins and their exchange dynamics in body fluids is difficult to assess. Here we measure the number of molecules adsorbed to sulfonate (PSOSO₃H) and carboxyl-(PSCOOH) polystyrene NPs using fluorescence correlation spectroscopy. We find that the fraction of molecules bound to NPs falls onto a single, universal adsorption curve, if plotted as a function of molar protein-to-NP ratio. The adsorption curve shows the build-up of a strongly bound monolayer up to the point of monolayer saturation (at a geometrically defined protein-to-NP ratio), beyond which a secondary, weakly bound layer is formed. While the first layer is irreversibly bound (hard corona), the secondary layer (soft corona) exhibits dynamic exchange, if competing unlabeled is added. In the presence of plasma proteins, the hard corona is stable, while the soft corona is almost completely removed. The existence of two distinct time scales in the protein off-kinetics, for both NP types studied here, indicates the possibility of an exposure memory effect in the NP corona

DCS measurements of Tf coated NP-corona complexes before and after incubation in plasma.

<p>A–C, apparent size comparison between Tf coated NPs dispersed in PBS, NPs incubated in plasma and isolated by spinning (so called hard corona) and NPs incubated in plasma <i>“in situ”</i> (so called full corona); D–F, the particle-corona complexes were isolated after either 5 min or 1 hour of incubation in the biological fluid. DCS experiment for the same NPs in PBS is reported for comparison in all graphs.</p

Strategies of PS NPs functionalization with Tf.

<p>Cartoon representation of 100 nm polystyrene NPs modified with sulphonated groups (PSOSO₃H, upper) and carboxylic groups (PSCOOH, down) coated with Tf by either physical adsorption (Tf@PSOSO₃H and <a href="mailto:Tf@PSCOOH" target="_blank">Tf@PSCOOH</a>) or covalent coupling (Tf-PSCOOH): A, pristine NPs; B, Tf coated NPs; C, Tf coated NPs after incubation in human plasma. Both Tf@PSOSO₃H and Tf-PSCOOH NPs are stable in human plasma, while Tf on <a href="mailto:Tf@PSCOOH" target="_blank">Tf@PSCOOH</a> NPs is easily replaced by environmental proteins.</p

Mass spectrometry data for human Tf amount on precoated PS nanoparticles before and after incubation in human plasma.

<p>Mass spectrometry data for human Tf amount on precoated PS nanoparticles before and after incubation in human plasma.</p