Development of Conjugated Gold Clusters for Studies on Cellular Internalization

金纳米颗粒作为药物传递和成像应用的平台已被广泛研究。它们通常可以以各种大小和形状合成，并且可以与各种分子（包括抗体，肽和药物）功能化。尽管世界各地的几个实验室仍在深入研究胶体金和量子点等较大纳米颗粒的细胞内化和分布，但由于在体外和细胞内用常规电子显微镜观察这些颗粒的困难，对超细（直径小于2纳米）纳米颗粒的吸收尚未得到很好的研究。我们合成了一种以对巯基苯甲酸（p-MBA）稳定的144个原子的超小金纳米粒子。这些纳米颗粒直径在2纳米以下，非常均匀，这是潜在的生物医学和纳米医学应用的理想特征。金（p-MBA）纳米颗粒的合成与谷胱甘肽（GSH）进行配体交换反应。我们使用两种强大的技术分析了所得的金（谷胱甘肽）纳米颗粒，这两种技术在纳米医学中应用的超小纳米颗粒的表征中尚未得到充分利用。定量扫描透射电子显微镜（STEM）成像显示，Au（GSH）具有高度均匀性，其核心金原子数（134）与母粒子Au（p-MBA）的144原子数几乎相同。超离心分析表明，Au（GSH）的水动力表观粒径分布较窄，为4.0 +/- 0.6 nm。接下来，将Au（GSH）以及与细胞穿透肽（TAT）结合的Au（GSH）复合物与HeLa细胞一起孵育，以评估纳米颗粒在细胞内的命运。STEM显示Au（GSH）和Au(GSH)-TAT都被细胞有效地内化并传递到细胞核。对图像的定量分析进一步表明，金（谷胱甘肽）以单个aunp的形式存在于细胞内部，也以含有2至10个单独纳米颗粒的小聚集体的形式存在。我们的结构方法提供了深入了解细胞穿透肽附着在模型金纳米颗粒上的内化机制，并揭示了缀合物在细胞内的命运。生物物理技术，包括STEM和分析超离心被用于研究具有不同簇大小和配体类型的超小金纳米颗粒与模型蛋白（如α -凝乳胰蛋白酶和溶菌酶）相互作用时的聚集行为。

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.