Understanding the nanoparticle-protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles

Abstract

Due to their small size, nanoparticles have distinct properties compared with the bulk form of the same materials. These properties are rapidly revolutionizing many areas of medicine and technology. Despite the remarkable speed of development of nanoscience, relatively little is known about the interaction of nanoscale objects with living systems. In a biological fluid, proteins associate with nanoparticles, and the amount and presentation of the proteins on the surface of the particles leads to an in vivo response. Proteins compete for the nanoparticle "surface," leading to a protein "corona" that largely defines the biological identity of the particle. Thus, knowledge of rates, affinities, and stoichiometries of protein association with, and dissociation from, nanoparticles is important for understanding the nature of the particle surface seen by the functional machinery of cells. Here we develop approaches to study these parameters and apply them to plasma and simple model systems, albumin and fibrinogen. A series of copolymer nanoparticles are used with variation of size and composition (hydrophobicity). We show that isothermal titration calorimetry is suitable for studying the affinity and stoichiometry of protein binding to nanoparticles. We determine the rates of protein association and dissociation using surface plasmon resonance technology with nanoparticles that are thiol-linked to gold, and through size exclusion chromatography of protein-nanoparticle mixtures. This method is less perturbing than centrifugation, and is developed into a systematic methodology to isolate nanoparticle-associated proteins. The kinetic and equilibrium binding properties depend on protein identity as well as particle surface characteristics and size.

Fig. 1.

ITC. Titration of HSA into solutions of 70 nm nanoparticles with 50:50 (Left) and 85:15 (Right) NIPAM/BAM in 10 mM Hepes/NaOH, 0.15 M NaCl, 1 mM EDTA, pH 7.5, is shown. (Upper) Raw data. (Lower) Integrated heats in each injection versus molar ratio of protein to nanoparticle together with a fit using a one site binding model (Eqs. 3–5 in SI Text). (Inset) Size comparison of albumin and particles of 70 or 200 nm diameter.

Fig. 2.

Size-exclusion chromatography (gel filtration) on a 1.5 × 95 cm sephacryl S1000 SF column. (A) Chromatogram of 200-nm 50:50 NIPAM/BAM particles alone. (B) SDS/PAGE of precipitated fractions from the elution of HSA with 200 nm 85:15 particles (Top), without (Middle) and with 200 nm 50:50 particles (Bottom). The arrow indicates the elution time of the particles (peak position).

Fig. 3.

SPR studies of plasma–nanoparticle interactions. (A) Cartoon of a gold surface with thiol-tethered particles and associated protein over which buffer is flown. (B and C) SPR data of plasma proteins injected at 60-fold dilution over 70-nm 85:15 NIPAM/BAM (blue) or 50:50 NIPAM/BAM (red) for 30 min (B) followed by buffer flow for 24 h (C, first 6,000 s shown). The black lines are computer fits using Eqs. 1 and 2 (SI Text).

Fig. 4.

Size exclusion chromatography of plasma proteins on a 1.5 × 95 cm sephacryl S1000 SF column. (Top Left) Cartoon showing the principle of the method where the darker protein has low affinity but high on and off-rate, and the smallest light gray protein has high affinity and lower dissociation rate. (Middle and Bottom Left) SDS/PAGE of precipitated plasma proteins from a chromatogram with 200 nm 50:50 NIPAM/BAM particles (Middle) and without particles (Bottom). Arrows indicate six protein bands migrating differently when particles are present. (Right) Chromatographic elution profiles of the particles (np), and proteins 1–6 and HSA alone (dashed line) and with nanoparticles (solid line). In 1–6, the y axis represents the density from the SDS/PAGE for each protein and fraction.

Fig. 5.

Centrifugation and size exclusion chromatography on isolated protein and particle complexes. (A) SDS/PAGE of proteins retrieved from nanoparticles in centrifugation experiments. The size and NIPAM/BAM ratio of the copolymer particles are given under the respective gel lanes. (B) Size-exclusion chromatogram of plasma protein associated with 200-nm 50:50 NIPAM/BAM particles. (C) SDS/PAGE on fractions 36–71.