A centrifugation-based physicochemical characterization method for the interaction between proteins and nanoparticles

Nanomedicine requires in-depth knowledge of nanoparticle–protein interactions. These interactions are studied with methods limited to large or fluorescently labelled nanoparticles as they rely on scattering or fluorescence-correlation signals. Here, we have developed a method based on analytical ultracentrifugation (AUC) as an absorbance-based, label-free tool to determine dissociation constants (KD), stoichiometry (Nmax), and Hill coefficient (n), for the association of bovine serum albumin (BSA) with gold nanoparticles. Absorption at 520 nm in AUC renders the measurements insensitive to unbound and aggregated proteins. Measurements remain accurate and do not become more challenging for small (sub-10 nm) nanoparticles. In AUC, frictional ratio analysis allows for the qualitative assessment of the shape of the analyte. Data suggests that small-nanoparticles/protein complexes significantly deviate from a spherical shape even at maximum coverage. We believe that this method could become one of the established approaches for the characterization of the interaction of (small) nanoparticles with proteins.

[1]  M. Shapiro,et al.  Direct determination of the orientation distribution function of cylindrical particles immersed in laminar and turbulent shear flows , 1991 .

[2]  Arthur J. Rowe,et al.  Analytical ultracentrifugation in biochemistry and polymer science , 1992 .

[3]  P. Schuck.  Sedimentation analysis of noninteracting and self-associating solutes using numerical solutions to the Lamm equation. , 1998, Biophysical journal.

[4]  G. Rivas,et al.  Characterization of heterologous protein-protein interactions using analytical ultracentrifugation. , 1999, Methods.

[5]  M. Ferrer,et al.  The conformation of serum albumin in solution: a combined phosphorescence depolarization-hydrodynamic modeling study. , 2001, Biophysical journal.

[6]  P. Schuck,et al.  Calculating sedimentation coefficient distributions by direct modeling of sedimentation velocity concentration profiles. , 2004, Methods in enzymology.

[7]  S. Franzen,et al.  Probing BSA binding to citrate-coated gold nanoparticles and surfaces. , 2005, Langmuir : the ACS journal of surfaces and colloids.

[8]  Borries Demeler,et al.  Modeling analytical ultracentrifugation experiments with an adaptive space-time finite element solution of the Lamm equation. , 2005, Biophysical journal.

[9]  J. Kimling,et al.  Turkevich method for gold nanoparticle synthesis revisited. , 2006, The journal of physical chemistry. B.

[10]  W. Maechtle.  Analytical Ultracentrifugation of Polymers and Nanoparticles , 2006 .

[11]  Sara Linse,et al.  Understanding the nanoparticle–protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles , 2007, Proceedings of the National Academy of Sciences.

[12]  Sara Linse,et al.  Detailed identification of plasma proteins adsorbed on copolymer nanoparticles. , 2007, Angewandte Chemie.

[13]  A. Lenhoff,et al.  Self-interaction nanoparticle spectroscopy: a nanoparticle-based protein interaction assay. , 2008, Journal of the American Chemical Society.

[14]  Francesco Stellacci,et al.  Water-soluble amphiphilic gold nanoparticles with structured ligand shells. , 2008, Chemical communications.

[15]  P. Brown,et al.  Characterizing Protein‐Protein Interactions by Sedimentation Velocity Analytical Ultracentrifugation , 2008, Current protocols in immunology.

[16]  Bin Zhang,et al.  Characterization of Protein Clusters of Diverse Magnetic Nanoparticles and Their Dynamic Interactions with Human Cells , 2009 .

[17]  Iseult Lynch,et al.  Protein-nanoparticle interactions: What does the cell see? , 2009, Nature nanotechnology.

[18]  Wolfgang J Parak,et al.  A quantitative fluorescence study of protein monolayer formation on colloidal nanoparticles. , 2009, Nature nanotechnology.

[19]  Jack F Douglas,et al.  Interaction of gold nanoparticles with common human blood proteins. , 2010, ACS nano.

[20]  Edward S Yeung,et al.  Separation of nanorods by density gradient centrifugation. , 2011, Journal of chromatography. A.

[21]  Jin Young Kim,et al.  Determination of nanoparticle size distribution together with density or molecular weight by 2D analytical ultracentrifugation , 2011, Nature communications.

[22]  Kenneth A. Dawson,et al.  Effects of the presence or absence of a protein corona on silica nanoparticle uptake and impact on cells. , 2012, ACS nano.

[23]  G. Nienhaus,et al.  Effect of protein adsorption on the fluorescence of ultrasmall gold nanoclusters. , 2012, Small.

[24]  Andrzej S Pitek,et al.  Reversible versus irreversible binding of transferrin to polystyrene nanoparticles: soft and hard corona. , 2012, ACS nano.

[25]  Marco P Monopoli,et al.  Biomolecular coronas provide the biological identity of nanosized materials. , 2012, Nature nanotechnology.

[26]  Alaaldin M. Alkilany,et al.  Nanoparticle-protein interactions: a thermodynamic and kinetic study of the adsorption of bovine serum albumin to gold nanoparticle surfaces. , 2013, Langmuir : the ACS journal of surfaces and colloids.

[27]  F. Stellacci,et al.  Electrical method to quantify nanoparticle interaction with lipid bilayers. , 2013, ACS nano.

[28]  Stefan Tenzer,et al.  Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology. , 2013, Nature nanotechnology.

[29]  L. Dai,et al.  Size‐ and Shape‐Dependent Fluorescence Quenching of Gold Nanoparticles on Perylene Dye , 2013 .

[30]  Teófilo Rojo,et al.  The challenge to relate the physicochemical properties of colloidal nanoparticles to their cytotoxicity. , 2013, Accounts of chemical research.

[31]  F. Stellacci,et al.  Effects of surface compositional and structural heterogeneity on nanoparticle-protein interactions: different protein configurations. , 2014, ACS nano.

[32]  F. Stellacci,et al.  Lipid tail protrusions mediate the insertion of nanoparticles into model cell membranes , 2014, Nature Communications.

[33]  Wolfgang J. Parak,et al.  Protein corona formation around nanoparticles – from the past to the future , 2014 .

[34]  W. Peukert,et al.  Multidimensional analysis of nanoparticles with highly disperse properties using multiwavelength analytical ultracentrifugation. , 2014, ACS nano.