Development of Conjugated Gold Clusters for Studies on Cellular Internalization

用于细胞内化研究的共轭金簇的开发

• 批准号: 8933888
• 负责人: Richard Leapman
• 金额: $ 6.07万
• 依托单位: NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8933888
• 关键词: Acids; Affect; Behavior; Binding; Caliber; Calorimetry; Cell Nucleus; Cell-Matrix Junction; Cells; Complex; Development; Drug Delivery Systems; Electron Microscopy; Fluorescence; Glutathione; Gold; Gold Colloid; Hela Cells; Image; Image Analysis; In Vitro; Incubated; Indium; Individual; Ligands; Light; Microtomy; Modeling; Muramidase; Parents; Peptide antibodies; Peptides; Pharmaceutical Preparations; Proteins; Quantum Dots; Reaction; Scanning Transmission Electron Microscopy Procedures; Shapes; Silver; Staining method; Stains; Techniques; Titrations; Variant; analytical ultracentrifugation; biophysical techniques; cell preparation; chymotrypsin A; in vivo; insight; light microscopy; nanomedicine; nanoparticle; particle; uptake

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.

金纳米颗粒作为药物输送和成像应用的平台已经得到了广泛的研究。它们通常可以被合成成各种大小和形状，并且可以用包括抗体、多肽和药物在内的各种分子来功能化。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，但对超小纳米颗粒(直径小于2 nm)的吸收还没有得到很好的研究，部分原因是很难用传统的电子显微镜在体外和细胞内观察这些颗粒。我们合成了一种144个原子的超小纳米金配体--对羟基苯甲酸(p-MBA)稳定的纳米金。这些纳米粒子的直径小于2 nm，并且非常均匀，这两个特点都是潜在的生物医学和纳米医学应用的理想特征。通过与谷胱甘肽(GSH)的配体交换反应合成了Au(p-MBA)纳米粒子。我们使用两种强大的技术分析了生成的Au(GSH)纳米颗粒，这两种技术在表征纳米医学中应用的超小纳米颗粒方面一直没有得到充分利用。定量扫描电子显微镜(STEM)成像显示，Au(GSH)具有高度的均一性，其核心金原子数(134)几乎与母体144原子的Au(p-MBA)纳米粒子相同。分析超速离心法表明，Au(GSH)的流体动力学表观尺寸分布较窄，为4.0+/-0.6 nm。接下来，将Au(GSH)以及Au(GSH)与细胞穿透肽(TAT)的络合物与HeLa细胞孵育，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输送到细胞核。对图像的定量分析进一步表明，Au(GSH)以单个AuNPs的形式存在于细胞内部，并以包含2至10个单独的纳米粒子的小聚集体的形式存在。我们的结构方法提供了对附着在模型金纳米颗粒上的穿透性细胞多肽的内化机制的洞察，以及对偶联物在细胞内的命运的揭示。包括STEM和分析超速离心法在内的生物物理技术正被用来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质如α-糜蛋白酶和溶菌酶相互作用时的聚集行为。