Ultrastructure of a Carbon Nanotube-based Delivery System for Cancer Therapy

用于癌症治疗的碳纳米管输送系统的超微结构

• 批准号: 8158003
• 负责人: Richard Leapman
• 金额: $ 4.06万
• 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/8158003
• 关键词: Adsorption; Binding; Carbon; Carbon Nanotubes; Cytoplasm; Data; Dehydration; Development; Electrons; Future; Glutaral; Head and Neck Squamous Cell Carcinoma; Image; Imagery; Incubated; Length; Malignant Epithelial Cell; Nanotubes; National Institute of Dental and Craniofacial Research; Pharmaceutical Preparations; Resolution; Scanning Transmission Electron Microscopy Procedures; System; base; cancer therapy; killings; malignant mouth neoplasm; sample fixation

Carbon nanotubes with attached drugs and directing ligands have excellent potential for targeted drug delivery. Single wall carbon nanotubes (SWNT) with very highly specific surface areas can be derivatized with biomolecules either through chemical attachment, adsorption or encapsulation. Such bioconjugates on SWNTs have the ability to deliver bioactive molecules across cell membranes and even into cell nuclei. We have performed ultrastructural experiments to characterize the nanobioconjugates and to investigate the application of functionalized carbon nanotubes to deliver therapeutic drugs to oral cancer cells. The anti-cancer drug cisplatin was covalently bound to single walled carbon nanotubes (SWNTs) and these were in turn bound to epidermal growth factor (EGF), which is expressed at very high levels by oral cancer cells. Characterization of such SWNT nanobioconjugates is important for the successful development of these functional bionanomaterials. We have applied atomic-scale scanning transmission electron microscopy (STEM) to visualize and quantitate single Pt-based drug molecules attached to single-wall carbon nanotubes designed for targeted drug-delivery. Annular dark-field STEM imaging enabled visualization of the first-line anticancer drug cisplatin on the nanotubes at single molecule levels. The identity and presence of cisplatin on the nanotubes were confirmed using energy-dispersive x-ray spectroscopy and Fourier transform infrared spectroscopy. STEM tomography was also used to provide additional information about the loading of cisplatin on the nanotube bioconjugates. Cultured head and neck squamous cell carcinoma (HNSCC) cells of epithelial origin incubated with the functionalized nanoparticles were prepared for electron microscopy by fixation in glutaraldehyde and osmium tetroxide, followed by dehydration and embedding in epon. Electron micrographs, recorded digitally using a 120 kV TEM, revealed that bundles of SWNTs had entered the cells. In most HNSCC cells the SWNT bundles were distributed throughout the cytoplasm, whereas some cells showed a higher concentration of nanotubes adjacent to the cell nucleus. The results confirmed lower resolution data obtained from confocal fluorescence microscopy but provided more detailed information about the subcellular structures associated with the nanotubes after entry. The ultrastructural data are being correlated with optical measurements and cell viability assays conducted in NIDCR. We have also used scanning transmission electron microscopy (STEM) and x-ray nanoanalysis to characterize individual nanotubes and to determine the distribution of bound platinum atoms. Characterization of these nanoparticle bioconjugates is essential for the future progress of this approach. In particular, nanotube size, and number of biomolecules per nanotube length are important parameters, especially for drug delivery. Our STEM results show for the first time atomic scale visualization and quantification of single Pt-based drug molecules attached to SWNTs.

带有附着药物和指导配体的碳纳米管具有靶向药物递送的巨大潜力。具有非常特异性表面区域的单壁碳纳米管（SWNT）可以通过化学附着，吸附或封装来衍生作用。 SWNT上的这种生物缀合物具有跨细胞膜甚至细胞核中传递生物活性分子的能力。我们已经进行了超微结构实验，以表征纳米偶联物，并研究功能化碳纳米管的应用以将治疗药物提供给口腔癌细胞。抗癌药物顺铂与单壁碳纳米管（SWNT）共价结合，而这些抗癌纳米管（SWNT）又与表皮生长因子（EGF）结合，该因子（EGF）通过口腔癌细胞在非常高的水平表达。 此类SWNT纳米二聚体的表征对于这些功能性bionanomatials的成功开发很重要。我们已经应用了原子尺度扫描透射电子显微镜（STEM）来可视化和定量与针对靶向药物交付的单壁碳纳米管相连的单个基于PT的药物分子。 环形暗场茎成像使单分子水平的纳米管上的一线抗癌药物顺铂具有可视化。 使用能量分散性X射线光谱和傅立叶变换红外光谱证实了顺铂在纳米管上的身份和存在。 STEM断层扫描还用于提供有关纳米管生物结合物上顺铂载素的其他信息。 与功能化纳米颗粒一起孵育的上皮起源的培养的头颈鳞状细胞癌（HNSCC）细胞通过固定在戊二醛和四氧化甲醛中，制备电子显微镜，然后在脱水中脱水并嵌入Epon中。使用120 kV TEM以数字方式记录的电子显微照片显示，SWNT束进入了细胞。在大多数HNSCC细胞中，SWNT束分布在整个细胞质中，而某些细胞显示出较高的与细胞核相邻的纳米管。结果证实了从共聚焦荧光显微镜获得的较低分辨率数据，但提供了有关进入后与纳米管相关的亚细胞结构的更详细信息。超微结构数据与NIDCR中进行的光学测量和细胞活力测定相关。我们还使用了扫描透射电子显微镜（Stem）和X射线纳米分析来表征单个纳米管，并确定结合铂原子的分布。这些纳米颗粒生物缀合物的表征对于这种方法的未来进步至关重要。特别是，纳米管大小和每纳米管长度的生物分子数量是重要的参数，尤其是用于药物输送。我们的STEM结果首次显示了原子量表可视化和对SWNT上附着的单个基于PT的药物分子的定量。