Structural Analysis of Low-Density Lipoprotein Interactions with Proteins and Corona Formation

Lipoproteins are naturally occurring biological nanoparticles circulating in our bloodstream. Researchers have paid great attention to their function in cholesterol transport and to their relationship with various diseases like cardiovascular disease and metabolic disorder. Their characteristics as nanoparticles have long been ignored. In a biological environment, lipoproteins may also bind with various proteins, and form hard and/or a soft corona on their surface, similar to other nanoparticles. In this thesis, low-density lipoproteins (LDL) are isolated using density gradient ultracentrifugation (DGUC), followed by fast protein liquid chromatography (FPLC) purification to minimize the presence of pre-existing protein corona. The purified LDL are characterized through dynamic light scattering (DLS) and transmission electron microscopy (TEM) before being incubated with human serum albumin (HSA), immunoglobulin G (IgG), and apo-transferrin to form a protein corona. These proteins were selected based on their varying abundance in plasma and their differing affinities for nanoparticle surfaces. HSA, the most abundant plasma protein, is primarily involved in molecular transport but interacts weakly with nanoparticles, a behaviour also observed for apo-transferrin. In contrast, IgG, the dominant immunoglobulin in blood, exhibits stronger binding to LDL. Sedimentation velocity analytical ultracentrifugation (SV-AUC) is employed to assess the size and interaction dynamics of LDL after corona formation. It is observed that the purity of the LDL samples significantly influences the interaction between LDL and proteins, indicating the existence of both a hard corona and a soft corona. Additionally, it was found that the binding of LDL to plasma proteins is highly protein-specific, with IgG forming stronger associations, leading to larger LDL-protein complexes. To further explore the function of protein corona on lipoproteins, isolated LDL is precipitated using a high concentration PEG solution. However, the results show no significant difference compared to LDL isolated through DGUC followed by FPLC, indicating that PEG precipitation does not alter LDL behaviour under these conditions. This thesis provides valuable insights into the interactions between lipoproteins and plasma proteins, contributing to a deeper understanding of protein corona formation on LDL. Furthermore, the findings highlight the potential to modulate protein corona composition, paving the way for advancements in lipoprotein-based therapeutics for precision medicine.