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.

附着有药物和定向配体的碳纳米管在靶向药物输送方面具有巨大的潜力。具有非常高比表面积的单壁碳纳米管（SWNT）可以通过化学附着、吸附或封装用生物分子衍生化。这种单壁碳纳米管上的生物结合物能够将生物活性分子穿过细胞膜，甚至进入细胞核。我们进行了超微结构实验来表征纳米生物缀合物，并研究功能化碳纳米管向口腔癌细胞输送治疗药物的应用。抗癌药物顺铂与单壁碳纳米管 (SWNT) 共价结合，而单壁碳纳米管又与表皮生长因子 (EGF) 结合，而表皮生长因子 (EGF) 在口腔癌细胞中以非常高的水平表达。 这种 SWNT 纳米生物共轭物的表征对于这些功能性生物纳米材料的成功开发非常重要。我们应用原子级扫描透射电子显微镜 (STEM) 对附着在单壁碳纳米管上的单个铂基药物分子进行可视化和定量，该单壁碳纳米管专为靶向药物输送而设计。 环形暗场 STEM 成像能够在单分子水平上对纳米管上的一线抗癌药物顺铂进行可视化。 使用能量色散 X 射线光谱和傅里叶变换红外光谱证实了纳米管上顺铂的身份和存在。 STEM 断层扫描还用于提供有关纳米管生物缀合物上顺铂负载的附加信息。 与功能化纳米颗粒一起孵育的培养的上皮来源的头颈鳞状细胞癌（HNSCC）细胞通过固定在戊二醛和四氧化锇中，然后脱水并包埋在epon中，准备用于电子显微镜检查。使用 120 kV TEM 数字记录的电子显微照片显示，单壁碳纳米管束已进入细胞。在大多数 HNSCC 细胞中，SWNT 束分布在整个细胞质中，而一些细胞在细胞核附近显示出更高浓度的纳米管。结果证实了从共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的铂原子的分布。这些纳米粒子生物共轭物的表征对于该方法的未来进展至关重要。特别是，纳米管尺寸和每纳米管长度的生物分子数量是重要参数，特别是对于药物输送而言。我们的 STEM 结果首次显示了附着在单壁碳纳米管上的单个铂基药物分子的原子尺度可视化和定量。