Mass Mapping of Macromolecular Assemblies

大分子组装体的质量作图

• 批准号: 7967858
• 负责人: Richard Leapman
• 金额: $ 4.07万
• 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/7967858
• 关键词: Acceleration; Acids; Binding; Blood Circulation; Caliber; Cells; Chelating Agents; Chemicals; Contrast Media; Dendrimers; Diagnosis; Disease; Drug Delivery Systems; Effectiveness; Electron Microscope; Electrons; Energy-Filtering Transmission Electron Microscopy; Generations; Goals; Guns; Half-Life; Image; Label; Laboratories; Ligands; Magnetic Resonance Imaging; Malignant Neoplasms; Maps; Measurement; Measures; Medical Imaging; Molecular; Molecular Weight; Nanostructures; Pharmaceutical Preparations; Physiological; Property; Proteins; Reagent; Scanning; Scanning Transmission Electron Microscopy Procedures; Shapes; Solutions; Source; Surface; Therapeutic Agents; Time; Tissues; Vascular Permeabilities; amino group; aqueous; design; detector; macromolecular assembly; molecular mass; nanocarrier; nanomedicine; nanoparticle; neoplastic; physical property; structural biology; transmission process; voltage

The scanning transmission electron microscope (STEM) has made significant contributions to structural biology by providing accurate determinations of the molecular masses of large protein assemblies that have arbitrary shapes and sizes. Nevertheless, STEM mass mapping has been implemented in very few laboratories, most of which have employed cold-field emission gun (FEG) electron sources operating at acceleration voltages of 100 kV and lower. Here we show that a 300 kV commercial transmission electron microscope (TEM) equipped with a thermally assisted Shottky FEG can provide accurate STEM mass measurements of nanoparticles that have potential for use in cancer theranostics. Polyamidoamines (PAMAMs) comprise a common class of dendrimers that are highly soluble in aqueous solution and can contain a high number of chemically active terminal amino groups. In particular, PAMAM dendrimers conjugated with Gd3+-diethylene triamine pentaacetic acid (DTPA) could provide useful magnetic resonance (MR) imaging contrast agents for neoplastic tissue. The effectiveness in the use of dendrimers for medical imaging and drug delivery applications can depend, among other factors, on their average diameter, average molecular weight, degree of conjugation with chelating agents, number of Gd atoms, and degree of monodispersity. For MR imaging and treatment of neoplastic tissue, for example, these variables can be expected to impact critical physiological properties of the dendrimers such as blood-circulation half-life, vascular permeability, and retention time within the tissue. Native, unconjugated dendrimers typically have well-defined molecular weights and high degree of monodispersity. However, after conjugation with Gd-DTPA chelates, some of the physical properties of the native dendrimers become less well-defined and are difficult to accurately quantify through direct measurements. We have used a combination of STEM mass mapping and energy-filtered transmission electron microscopy (EFTEM) to characterize specific generations (G5, G6, G7) of PAMAM dendrimers. STEM images provide the distribution of molecular weights of the diffferent dendrimer generations, whereas EFTEM imaging at the Gd N45 excitation edge provides the numbers of bound Gd atoms. This approach provides a quantitative assessment of the degree of monodispersity of nanoparticles, which will be useful in developing these reagents for eventual applications in nanomedicine.

扫描透射电子显微镜 (STEM) 通过准确测定具有任意形状和大小的大型蛋白质组装体的分子质量，为结构生物学做出了重大贡献。 尽管如此，STEM 质量测绘在极少数实验室中得到实施，其中大多数实验室都采用在 100 kV 及更低加速电压下运行的冷场发射枪 (FEG) 电子源。在这里，我们展示了配备热辅助肖特基 FEG 的 300 kV 商用透射电子显微镜 (TEM) 可以提供纳米粒子的精确 STEM 质量测量，这些纳米粒子有可能用于癌症治疗诊断。 聚酰胺胺 (PAMAM) 包含一类常见的树枝状聚合物，它们在水溶液中高度可溶，并且可以含有大量化学活性末端氨基。 特别是，与 Gd3+-二亚乙基三胺五乙酸 (DTPA) 缀合的 PAMAM 树枝状大分子可以为肿瘤组织提供有用的磁共振 (MR) 成像造影剂。 树枝状聚合物在医学成像和药物输送应用中的有效性取决于其平均直径、平均分子量、与螯合剂的缀合程度、Gd 原子数量和单分散程度等因素。 例如，对于肿瘤组织的 MR 成像和治疗，这些变量预计会影响树枝状聚合物的关键生理特性，例如血液循环半衰期、血管通透性和组织内的保留时间。 天然的、非共轭的树枝状聚合物通常具有明确的分子量和高度的单分散性。 然而，与 Gd-DTPA 螯合物缀合后，天然树枝状聚合物的一些物理性质变得不太明确，并且难以通过直接测量准确量化。 我们结合使用 STEM 质量图和能量过滤透射电子显微镜 (EFTEM) 来表征 PAMAM 树枝状聚合物的特定代（G5、G6、G7）。 STEM 图像提供了不同代树枝状聚合物的分子量分布，而 Gd N45 激发边缘的 EFTEM 成像提供了结合的 Gd 原子的数量。 这种方法提供了纳米颗粒单分散程度的定量评估，这将有助于开发这些试剂以最终应用于纳米医学。