The angiotensin II receptor type 1 (AT1R) is a G protein-coupled receptor of paramount significance since it is overexpressed in a number of diseased tissues that are highly attractive for nanoparticle targeting. However, it is also expressed at physiological levels in healthy tissue. Multivalent interactions mediated by multiple AT1R-binding moieties per nanoparticle could promote a high binding avidity to AT1R overexpressing cells and concomitantly spare off-target tissue. To investigate the feasibility of this approach, angiotensin II was thiolated and conjugated to PEGylated quantum dots. Nanoparticle binding, uptake and affinity to several cell lines was investigated in detail. The colloids were rapidly taken up by clathrin-mediated endocytosis into AT1R-expressing cells and showed no interaction with receptor negative cells. The EC50 of the thiolated angiotensin II was determined to be 261 nM, whereas the ligand-conjugated Qdots activated the receptor with an EC50 of 8.9 nM. This 30-fold higher affinity of the nanoparticles compared to the unconjugated peptide clearly demonstrated the presence of multivalent effects when using agonist-targeted nanoparticles. Our study provides compelling evidence that, despite being immediately endocytosed, Ang II-coupled nanoparticles exert potent multivalent ligand-receptor interactions that can be used to establish high affinities to an AT1R overexpressing cell and tissue.
Keywords: Agonist; GPCR; ligand–receptor interaction; multivalency; multivalent enhancement; polyvalency; quantum dots; uptake.