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

Abstract

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.

Nanoparticle Characterization: What to Measure?

“…The extremely high sensitivity of AUC to variations in density and mass of the nanoparticles renders this technique particularly suitable for investigating particle surface coverage . Furthermore, the possibility of integrating multiwavelength optical characterization during sample sedimentation largely expands the range of application of AUC in nanoparticle characterization, enabling, e.g., to investigate nanoparticle‐protein interaction or particle shape and optical behavior of polydisperse sample populations in a single experiment …”

Conformational-transited protein corona regulated cell-membrane penetration and induced cytotoxicity of ultrasmall Au nanoparticles

“…[1][2][3][4][5] During the past decade, substantial progress has been made in understanding the physicochemical properties of the protein corona. 2,[5][6][7][8][9][10][11][12][13][14][15][16][17] Ultrasmall metal nanoparticles of core size <3 nm, sometimes presenting unique electrical and optical properties, have received enormous attention recently. [18][19][20][21] The development of ultrasmall gold nanoparticles (AuNPs) provides an avenue for cancer therapy and in other nanomedicine elds.…”

Regimes of Biomolecular Ultrasmall Nanoparticle Interactions

“…Analytical ultracentrifugation (AUC) and fluorescence correlation spectroscopy (FCS) are very useful and sophisticated techniques that have been successfully employed to investigate USNP-protein interactions,m ainly using single proteins. [12] We used as eries of gel assays for in situ investigation of biomolecules-USNPs interactions. [13] TheGNP-PEG-COOH samples (5 nm, 3nma nd 2nm) were normalized by surface area and exposed to human plasma (HP).…”