Gold nanoparticles have been widely investigated as platforms for drug delivery and imaging applications. They can be typically synthesized in a wide range of sizes and shapes, and can be functionalized with a variety of molecules including antibodies, peptides, and drugs. Whereas the cellular internalization and distribution of larger nanoparticles such as colloidal gold and quantum dots continues to be intensively investigated by several labs around the world, the uptake of ultrasmall (diameter less than 2 nm) nanoparticles has not been as well studied, due in part to difficulties in visualizing these particles both in vitro and inside cells by conventional electron microscopy. We have synthesized an ultrasmall, 144-atom gold nanoparticle ligand-stabilized with para-mercaptobenzoic acid (p-MBA). These nanoparticles are under 2 nm in diameter and extremely uniform, which are both desirable features in potential biomedical and nanomedicine applications. The synthesis of Au( p-MBA) nanoparticles was followed by a ligand exchange reaction with glutathione (GSH). We analyzed the resulting Au(GSH) nanoparticles using two powerful techniques that have been underutilized in the characterization of ultrasmall nanoparticles for applications in nanomedicine. Quantitative scanning transmission electron microscopy (STEM) imaging revealed that Au(GSH) was highly uniform and had almost the same number of core gold atoms (134) as the parent 144-atom Au( p-MBA) nanoparticle. Analytical ultracentrifugation showed that Au(GSH) had a narrow hydrodynamic apparent size distribution of 4.0 +/- 0.6 nm. Next, Au(GSH) as well as complexes of Au(GSH) bound to the cell-penetrating peptide (TAT) were incubated with HeLa cells to evaluate the intracellular fate of the nanoparticles. STEM revealed that both Au(GSH) and Au(GSH)-TAT were effectively internalized by the cells and delivered to the nucleus. A quantitative analysis of the images further indicated that Au(GSH) were present in the cell interior as single AuNPs as well as in the form of small aggregates containing from 2 to 10 individual nanoparticles. Our structural approach provides insight into the mechanisms of internalization of cell-penetrating peptides attached to model gold nanoparticles, as well as shed light on the intracellular fate of the conjugates. Biophysical techniques, including STEM and analytical ultracentrifugation are being used to investigate the aggregation behavior of ultrasmall gold nanoparticles with different cluster size and ligand type, when they interact with model proteins such as alpha-chymotrypsin and lysozyme.
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