Methodology Development for Understanding Interactions at the Nano-bio Interface
- Author(s): Zeng, Shang
- Advisor(s): Zhong, Wenwan
- et al.
Nanoparticles have attracted huge attention due to their unique size-dependent properties. The fabrication of nanoparticles and exploration of their potential use has become a strong interest in numerous areas including biomedicine and information technology. These highly potential applications of nanoparticles lead to the concern for the design of safe nano-biomaterials and promoted the study of nanotoxicology, and safety assessment research in parallel to development of applications.
Protein adsorption is believed to be the primary event when nanoparticles enter a living system. In this dissertation, focus was placed on method development for evaluating the nanoparticle interactions with either a single protein or a cellular proteome. For the nanoparticle-single protein interaction, a novel method was developed for quantitative measurement on nanoparticle-protein interaction using capillary electrophoresis. With this method, we screened the affinity of polyacrylic acid-coated nanoparticles towards a selected list of proteins and found that protein adsorption was so sensitive to the surface properties of particles that it can reveal even small variations in the structure of a nanoparticle surface ligand. The binding site of nanoparticles on protein was also revealed by cross-linking chemistry coupled with mass spectrometry. The peptides located at protein-nanoparticle interface were cross-linked to nanoparticle surface, cleaved off after trypsin digestion and identified by mass spectrometry.
For the nanoparticle-proteome interaction, we believe for nanoparticles to work effectively in a biological system, an appropriate protein corona is needed for their cell internalization and cellular response. CpG-conjugated single wall carbon nanotubes were found to have superior anti-tumor efficacy than free CpG in glioma-bearing mice. Therefore we analyzed the protein corona composition of CpG-conjugated single wall carbon nanotubes in mouse macrophage cells. The CpG-conjugated single wall carbon nanotubes were incubated with cell lysate or live cells, then isolated and processed with protease. The resulted peptides were analyzed with liquid chromatography-mass spectrometry. The abundance of proteins was determined by calculating their exponentially modified protein abundance index. We found 35 proteins were specifically selected and enriched by CpG motifs. Their confirmation change or function inhibition may potentially contribute to the excellent anti-tumor efficacy of CpG functionalized single wall carbon nanotubes.