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

碳纳米管与药物和定向配体的连接具有良好的靶向给药潜力。单壁碳纳米管（SWNT）具有非常高的比表面积，可以通过化学附着、吸附或包封与生物分子衍生。单壁碳纳米管上的这种生物缀合物具有将生物活性分子递送穿过细胞膜甚至进入细胞核的能力。我们已经进行了超微结构实验，以表征纳米生物缀合物，并研究功能化碳纳米管的应用，以提供治疗药物的口腔癌细胞。抗癌药物顺铂与单壁碳纳米管（SWNT）共价结合，这些碳纳米管又与表皮生长因子（EGF）结合，后者在口腔癌细胞中以非常高的水平表达。 这种单壁碳纳米管纳米生物复合物的表征对于这些功能性生物纳米材料的成功开发是重要的。我们已经应用了原子级扫描透射电子显微镜（STEM）可视化和定量的单Pt为基础的药物分子连接到单壁碳纳米管设计的靶向药物输送。 环形暗场STEM成像使一线抗癌药物顺铂在单分子水平上在纳米管上可视化。 使用能量色散X射线光谱和傅里叶变换红外光谱的身份和存在的顺铂的纳米管进行了确认。 STEM断层扫描也用于提供关于顺铂在纳米管生物缀合物上的负载的额外信息。 通过在戊二醛和四氧化锇中固定，然后脱水并包埋在epon中，制备与功能化纳米颗粒孵育的上皮来源的培养的头颈部鳞状细胞癌（HNSCC）细胞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，成束的SWNT已经进入细胞。在大多数HNSCC细胞中，SWNT束分布在整个细胞质中，而一些细胞显示出较高浓度的纳米管邻近细胞核。结果证实了从共聚焦荧光显微镜获得的较低分辨率的数据，但提供了更详细的信息与纳米管进入后的亚细胞结构。超微结构的数据正在与光学测量和细胞活力测定进行NIDCR。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，并确定结合铂原子的分布。这些纳米颗粒生物共轭物的表征是这种方法的未来进展所必需的。特别地，纳米管尺寸和每纳米管长度的生物分子数量是重要的参数，尤其是对于药物递送。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。

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.