Development of Conjugated Gold Clusters for Studies on Cellular Internalization

金纳米颗粒作为药物输送和成像应用的平台已被广泛研究。 它们通常可以以广泛的尺寸和形状合成，并且可以用各种分子（包括抗体、肽和药物）官能化。 尽管世界各地的几个实验室继续深入研究较大纳米颗粒（如胶体金和量子点）的细胞内化和分布，但超小（直径小于2 nm）纳米颗粒的摄取尚未得到充分研究，部分原因是难以通过常规电子显微镜在体外和细胞内观察这些颗粒。 我们已经合成了一个超小的，144个原子的金纳米粒子配体稳定与对巯基苯甲酸（p-MBA）。这些纳米颗粒的直径小于2nm并且非常均匀，这在潜在的生物医学和纳米医学应用中都是理想的特征。 Au（p-MBA）纳米颗粒的合成之后是与谷胱甘肽（GSH）的配体交换反应。我们分析了所得的Au（GSH）纳米粒子，使用两个强大的技术，已未充分利用的超小纳米粒子在纳米医学中的应用程序的特性。定量扫描透射电子显微镜（STEM）成像显示，Au（GSH）是高度均匀的，并且具有与母体144个原子的Au（p-MBA）纳米颗粒几乎相同数量的核心金原子（134）。超离心分析表明，Au（GSH）具有4.0 +/- 0.6 nm的窄流体动力学表观粒径分布。接下来，将结合至细胞穿透肽（达特）的Au（GSH）以及Au（GSH）的复合物与HeLa细胞一起孵育以评估纳米颗粒的细胞内命运。STEM显示Au（GSH）和Au（GSH）-达特均被细胞有效内化并递送至细胞核。图像的定量分析进一步表明，Au（GSH）作为单个AuNP以及以含有2至10个单独的纳米颗粒的小聚集体的形式存在于细胞内部。我们的结构方法提供了深入了解附着到模型金纳米颗粒的细胞穿透肽的内化机制，以及阐明缀合物的细胞内命运。生物物理技术，包括STEM和分析超离心被用来研究具有不同簇大小和配体类型的超小金纳米粒子的聚集行为，当它们与模型蛋白质如α-糜蛋白酶和溶菌酶相互作用时。

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.