Gold nanoparticles (Au NPs) have attracted much interest in biological applications due to their excellent optoelectronic properties and ease of surface functionalization. However, while Au NPs are positioned to revolutionize nanotechnology based biomedical applications, our fundamental understanding at the nanoparticle-biomolecular interface and its resultant impact on cells is still limited. In particular, analysis of the spatial arrangement of nanoparticle’s surface ligands using scanning tunneling microscopy is a highly controversial topic. Addition of nanoparticles to cell culture media was shown to result in a hard and soft protein corona formation, which acted to mitigate/reduce the proposed chemical capabilities of nanoparticles. More critically but even less well understood is the precise orientation of proteins upon adsorption as well as the possible change in protein’s conformation, which can alter the protein’s intrinsic function. This dissertation will thus focus on developing our understanding at this interface, by probing the chemistries at this nanoparticle-biomolecular interface and subsequently, how the Au NPs influence cellular responses.The question of spatial location of different ligands on nanoparticle surfaces with diameters less than 100 nm is an important one that is difficult to quantitatively address. To investigate the spatial arrangement of biomolecules on Au NPs, the surface of 20, 50, and 90 nm Au NPs were functionalized with two different lipids, both single and mixed, using two different surface chemical procedures utilizing electrostatic or hydrophobic interactions. Mass spectrometry supported the presence of both lipids in the mixed-lipid systems on nanoparticles, and it was observed that the surface chemistry of Au NPs influenced the relative ratios of mixed lipids incorporated. Electron microscopy evidence showed domain sizes for one lipid apparently a quarter to a half the projected diameter for 50 and 90 nm particles; but for 20 nm particles, there is no evidence for the existence of patches of the two lipids.To study the potential use of Au NPs to limit α-synuclein (α-syn) misfolding, the binding and orientation of α-syn on anionic and cationic Au NPs were investigated. On anionic Au NPs, α-syn was determined to interact with 20 and 90 nm Au NPs via multilayered adsorption, consisting of a strong electrostatic interaction between α-syn and Au NPs in the hard corona and a weaker noncovalent protein−protein interaction in the soft corona. On cationic PAH Au NPs, titration of α-syn into cationic Au NP at >2000 α-syn/cationic Au NP caused the flocculation and sedimentation of α-syn coated PAH Au NPs. The orientation of α-syn onto Au NPs was studied using protease digestion method, revealing that α-syn absorbs onto anionic Au NPs via its N-terminus while on cationic Au NPs, a random orientation of α-syn was adopted. Comparison of the digestion pattern of α-syn on both Au NP with respect to free α-syn reveal an increase in the release of peptides from the N-terminus (amino acid 1–23, lysine position 10) and a decreased number of peptides in the non-amyloid component region (amino acid 59–97, lysine position 80) when adsorbed onto Au NPs, suggesting that the adsorption and binding orientation of α-syn depends on the surface charge of Au NPs.The aggregation of Au NPs in cell media is a common phenomenon that can influence NP-cell interactions. This interaction can be more precisely controlled by the formation of a protein corona on Au NPs before introduction into a high salt media. Cell viability assays showed that non-aggregated Au NPs were less toxic than their aggregated counterparts in human dermal fibroblast (HDF) cells. Fluorescence confocal imaging demonstrated that cellular F-actin fiber formation was less disrupted with non-aggregated Au NPs.
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Studies at the nanoparticle-biomolecular interface and beyond
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