Silica Nanoparticle–Protein Aggregation and Protein Corona Formation Investigated with Scattering Techniques

Protein–nanoparticle interactions and the resulting corona formation play crucial roles in the behavior and functionality of nanoparticles in biological environments. In this study, we present a comprehensive analysis of protein corona formation with superfolder green fluorescent protein (sfGFP) and bovine serum albumin in silica nanoparticle dispersions using small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS). For the first time, we subtracted the scattering of individual proteins in solution and individual nanoparticles from the protein–nanoparticle complexes. This approach effectively isolated the contributions of specific components within the corona. Our form factor analysis revealed consistent core–shell sphere thicknesses but varied attractive interaction strengths of the nanoparticle complexes, influenced by the protein corona and the surface properties of silica and aminated silica nanoparticles. Interestingly, fractal analysis of nanoparticles showed a transition from surface to mass fractals for sfGFP samples at high protein:nanoparticle molar ratios of over 264,000:1. DLS analysis highlighted aggregation behaviors, including the increasing size of protein–nanoparticle complexes and significant aggregation of both free proteins and complexes at ∼264,000 molar ratio. Large polydispersity and heterogeneous protein aggregation were observed at these high molar ratios. Both SAXS and DLS revealed transitions and changes in protein–nanoparticle interactions at molar ratios of 4000 to 44,000, consistent with corona formation, while pronounced aggregation was observed at a molar ratio of ∼264,000. These findings advance our understanding of the structural complexities in protein–nanoparticle association and suggest further avenues for refining characterization techniques in protein corona research.