(A) Schematic representation of NTCP-targeted nanoparticle binding to hepatocytes. Circulating nanoparticles pass the fenestrae of liver sinusoidal endothelial cells (LSEC) and subsequently bind to the NTCP in the basolateral membrane of hepatocytes facing the space of Disse. Prior to Myr-preS2-31 mediated NTCP binding, the myristoyl chain is inserted into the lipid bilayer. In close proximity to hepatocytes, acyl chain switches into cellular membrane due to high affinity of essential peptide sequence to NTCP binding site, thereby consolidating target transporter binding. (B) Nanoparticles were modified with 0.25 mol% of Myr-preS2-31 and labeled with radioactive 111In and fluorescent membrane dye (DiI, red signal). Planar imaging of mice 1 h post-injection. Competitive inhibition of liver binding by co-injection of free Myrcludex B clearly demonstrates NTCP-specific binding. Positions of liver and spleen are indicated by small circles. (C) Immunofluorescence staining of nanoparticles (red signal), targeting ligand (Myr-preS2-31, green signal, antibody staining), and Slc10a1 (cyan signal, antibody staining) in liver cryosections. Nuclei staining (blue signal) served as control for complete internalization; no overlap with Slc10a1. Scale bar = 20 µm. Colocalization analysis with Slc10a1 is represented by Pearson´s Correlation Coefficient (PCC). All values are shown as box plots of biological replicates (n = 3 independent experiments). *p<0.05. Numerical data for all graphs are shown in Figure 6—source data 1.