Gold labeled low density lipoprotein nanoparticles: Imaging lipoprotein biointeractions with CT, TEM and fluorescence

Introduction Low density lipoprotein (LDL) plays a critical role in cholesterol transport to peripheral tissues expressing the LDL receptor (LDLr). Supply of cholesterol is crucial for the maintenance of cell membranes. Furthermore, LDL is closely linked to the formation of atherosclerotic plaques and tumor growth. Gold nanoparticles have been used in many applications in nanomedicine, where they have been applied in radiotherapy, targeted drug delivery, and laser irradiation. In addition, gold nanoparticles have a major role in imaging and are used from transmission electron microscopy (TEM) to optical imaging, and as computed tomography (CT) contrast agents. Hence to be able to load LDL with gold nanoparticles would be extremely useful, allowing LDL to be tracked using various imaging modalities or to deliver therapeutics to diseased tissue. In this study, we developed a novel method to label LDL with gold nanocrystals and Cy5.5 (Au-LDL) (Figure 1A). This enables studying LDL interactions using a variety of imaging techniques, including CT, TEM, and fluorescence techniques. Methods and results Human LDL was sonicated in water for 5 min with 3 nm gold nanocores in a 1 mg ApoB100 to 2.8 mg gold ratio and purified via centrifugation. TEM confirmed that 77% of LDL was labeled with gold and showed a similar morphology and size to native LDL (Figure 1B,C), proving it vastly more effective than the well established Krieger substitution method (Figure S1A). Gel electrophoresis and Western blotting of LDL and Au-LDL had similar results (Figure S1B). In vitro studies were performed in LDLr expressing cells (HepG2, J774A.1, and B16-F10). Competition inhibition assays showed saturable uptake by fluorescence imaging (Figure 1D,E), CT (Figure S1C), and TEM (Figure S1E-M). B16-F10 tumor bearing mice were injected with LDL, Au-LDL and an Au-LDL resembling nanoemulsion lacking ApoB100 (Au-NE). CT showed a higher attenuation in the tumor tissue of mice injected with Au-LDL (29.3 ± 13.4 HU) or Au-NE (14.3 ± 12.0 HU) mice than uninjected mice (Figure S1D). Spectral CT analysis confirmed gold accumulation at the rim of the B16-F10 tumor in mice injected with Au-LDL (Figure 1F). Furthermore, TEM of B16-F10 tumor tissue showed gold uptake in vesicles of tumor cells, endothelial cells and macrophages (Figure 1G), which was also confirmed by FACS analysis. Identification of gold particles in TEM images was confirmed by EDX analysis. Discussion and conclusion In this study, we developed a novel method that allowed labeling of native human LDL with 3 nm gold nanocrystals, without altering LDL characteristics. Therefore, Au-LDL can likely be used as a marker to study LDL interactions, cholesterol metabolism, atherosclerotic plaque formation and tumor growth, with a variety of imaging techniques. In the future LDL could be used as a natural carrier platform to deliver gold nanomaterials and lipophilic drugs for therapeutic purposes. Clinical relevance Au-LDL could be an important new step in studying the cholesterol metabolism and behavior of LDL in a variety of diseases. Furthermore, natural LDL is a potent and promising carrier platform in nanomedicine. (Figure Presented).