Ultrasmall Gold Nanoparticles Decorated with Biomolecules for Specific Interaction with Cells and Proteins

Ultrasmall gold nanoparticles (diameter 1-2 nm) are smaller than most proteins and therefore well suited to elucidate particle-protein interactions after suitable surface functionalization. siRNA and fluorescing dyes (e.g. FAM, Cy5, Alexa647) were attached by copper-catalyzed azide-alkyne cycloaddition (click chemistry) to azide-terminated gold nanoparticles. Peptides of different length were directly attached via cysteine, taking advantage of the strong gold-sulfur bond. The particles were characterized by high-resolution transmission electron microscopy, differential centrifugal sedimentation, and ultraviolet spectroscopy. Extensive NMR spectroscopic investigations of dispersed particles were possible due to the ultrasmall nature of the particles. By combining the results of all these analytical techniques, a full image of the particles was gained, including the number of ligands (typically 5-10 per nanoparticle) and the details of the ligand-gold bond.<br/>The nanoparticles were surface-decorated with biomolecules and synthetic receptor molecules to interact with the surface of model proteins as probed by NMR spectroscopy, isothermal titration calorimetry, and fluorescence polarisation. Such particles are easily taken up by cells as demonstrated in classical two-dimensional cell cultures and in three-dimensional organoids. Notably, the particles were able to enter the cell nucleus and also to cross the blood-brain barrier in brain organoids that exhibit a functional blood-brain barrier in vitro. We also demonstrated how they were taken up by bacteria.<br/>In conclusion, covalently functionalized ultrasmall gold nanoparticles have a strong potential for specific interaction with proteins, for imaging and as drug carrier into cells and organoids.