Lipoprotein particles (LPs) are excellent transporters and have been intensively studied in cardiovascular diseases, especially regarding parameters such as their class distribution and accumulation, site-specific delivery, cellular internalization, and escape from endo/lysosomal compartments. The aim of the present work is the hydrophilic cargo loading of LPs. As an exemplary proof-of-principle showcase, the glucose metabolism-regulating hormone, insulin, was successfully incorporated into high-density lipoprotein (HDL) particles. The incorporation was studied and verified to be successful using Atomic Force Microscopy (AFM) and Fluorescence Microscopy (FM). Single-molecule-sensitive FM together with confocal imaging visualized the membrane interaction of single, insulin-loaded HDL particles and the subsequent cellular translocation of glucose transporter type 4 (Glut4).