Peptide-Conjugated Ultrasmall Gold Nanoparticles (2 nm) for Selective Protein Targeting
Tatjana Ruks, Κateryna Loza, Marc Heggen, Oleg Prymak, André Luiz Sehnem, Cristiano L. P. Oliveira, Peter Bayer, Christine Beuck, Matthias Epple
Abstract
Ultrasmall gold nanoparticles with a metallic core diameter of 2 nm were surface-conjugated with peptides that selectively target epitopes on the surface of the WW domain of the model protein hPin1 (hPin1-WW). The binding to the gold surface was accomplished via the thiol group of a terminal cysteine. The particles were analyzed by NMR spectroscopy, high-resolution transmission electron microscopy, and differential centrifugal sedimentation. The surface loading was determined by conjugating a FAM-labeled peptide, followed by UV–vis spectroscopy, and by quantitative 1H NMR spectroscopy, showing about 150 peptide molecules conjugated to each nanoparticle. The interaction between the peptide-decorated nanoparticles with hPin1-WW was probed by 1H–15N-HSQC NMR titration, fluorescence polarization spectroscopy (FP), and isothermal titration calorimetry (ITC). The particles showed a similar binding (KD = 10–20 μM) compared to the dissolved peptides (KD = 10–30 μM). Small-angle X-ray scattering (SAXS) showed that the particles were well dispersed and did not agglomerate after the addition of hPin1-WW (no cross-linking by the protein). Each nanoparticle was able to bind about 20 hPin1-WW protein molecules. An unspecific interaction with hPin1 was excluded by the attachment of a nonbinding peptide to the nanoparticle surface. The uptake by cells was studied by confocal laser scanning microscopy. The peptide-functionalized nanoparticles penetrated the cell membrane and were located in the cytosol. In contrast, the dissolved peptide did not cross the cell membrane. Peptide-functionalized nanoparticles are promising agents to target proteins inside cells.