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.

金纳米颗粒已被广泛研究为药物输送和成像应用的平台。 它们通常可以在各种尺寸和形状的范围内合成，并且可以用多种分子功能化，包括抗体，肽和药物。 尽管世界各地的几个实验室继续对较大纳米颗粒（例如胶体金和量子点）的细胞内在化和分布进行深入研究，但对超大颗粒的吸收（直径小于2 nm）的纳米颗粒的吸收尚未得到很好的研究，这是由于这些粒子在体外和公会上的难度都没有得到很好的研究，因此，这些粒子的困难是在公导中的范围。 我们已经合成了用Para-Mercaptobenzoic Acid（P-MBA）稳定的144个原子金纳米颗粒配体。这些纳米颗粒的直径低于2 nm，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。我们使用两种功能强大的技术分析了所得的AU（GSH）纳米颗粒，这些技术在超质纳米颗粒中用于纳米医学中应用的表征不足。定量扫描透射电子显微镜（STEM）成像表明，AU（GSH）高度均匀，几乎具有与父144-ATOM AU（P-MBA）纳米粒子相同数量的核心金原子（134）。分析性超速离心表明，AU（GSH）的流体动力学尺寸分布为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）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表明，AU（GSH）和AU（GSH）-TAT都被细胞有效地内化并传递到细胞核中。对图像的定量分析进一步表明，在细胞内部以及包含2至10个单个纳米颗粒的小聚集体的形式中，AU（GSH）存在于单个AUNP中。我们的结构方法提供了有关连接到模型金纳米颗粒的细胞穿透肽内在化机制的洞察力，以及对结合物的细胞内命运的启示。当生物物理技术（包括茎和分析性超速离心）用于研究具有不同簇大小和配体类型的超质金纳米颗粒的聚集行为，当它们与α-苯甲酸胰蛋白酶和溶菌酶等模型蛋白相互作用时。