Comparative Analysis of Lipoprotein Purification Techniques and Their Impact on Corona Formation

The protein corona, caused by nanoparticle-protein interaction on the surface of nanoparticles, has long been studied due to its ability to modulate the particle’s biological properties. It is a dynamic layer of proteins that adsorb onto nanoparticles, influencing their structural characteristics and functions. There are two types of protein corona: hard corona, composed of tightly bound and hardly dissociable proteins, and soft corona, composed of weakly bound proteins with more frequent exchange. Lipoproteins, whose size ranges from 10 to 1000 nm, are an important family of nanoparticles; nevertheless, the study of the protein corona on lipoproteins remains largely unexplored. This thesis aims to investigate the protein corona composition of low-density lipoproteins (LDL) isolated through different extraction protocols and purification methods. Density gradient ultracentrifugation (DGUC), polyethylene glycol (PEG) precipitation, fast protein liquid chromatography (FPLC) and various combinations of them were applied. The thesis focuses on evaluating how the isolation process influences the adsorption of serum proteins onto LDL, causing variations in the existence of hard corona and soft corona, which are reflected by their physical properties and protein composition. Different isolation approaches result in LDL with different characteristics. Notably, DGUC has been observed to be particularly effective at removing the soft corona, where LDL extracted with DGUC are smaller. Indeed, these LDL also compose of much fewer numbers of proteins. This is likely due to the high centrifugal force and density separation that strip proteins from LDL surface during the isolation. In comparison, LDL extracted with merely FPLC (or PEG precipitation followed by FPLC) can keep more corona with them, resulting in larger size. More proteins were also detected from those LDL in the proteomics analysis. These findings emphasize the importance of lipoprotein-protein interaction and corona on lipoproteins. By leveraging the nano-bio interaction through selecting suitable isolation protocols, the protein corona on lipoproteins can be regulated, paving the way for lipoprotein-based disease detection and therapy.