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

碳纳米管具有附着药物和定向配体，具有良好的靶向药物递送潜力。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、吸附或包封与生物分子衍生化。这种单壁碳纳米管上的生物偶联物具有将生物活性分子传递穿过细胞膜甚至进入细胞核的能力。我们进行了超微结构实验来表征纳米生物偶联物，并研究功能化碳纳米管在向口腔癌细胞输送治疗药物中的应用。抗癌药物顺铂与单壁碳纳米管（SWNTs）共价结合，而这些碳纳米管又与表皮生长因子（EGF）结合，而表皮生长因子在口腔癌细胞中表达得非常高。表征这些纳米SWNT生物偶联物对于成功开发这些功能生物纳米材料非常重要。我们已经应用原子尺度扫描透射电子显微镜（STEM）对附着在单壁碳纳米管上的单个基于pt的药物分子进行了可视化和定量分析。环形暗场STEM成像使一线抗癌药物顺铂在纳米管上的单分子水平可视化。利用能量色散x射线光谱和傅里叶变换红外光谱证实了顺铂在纳米管上的存在。STEM断层扫描也用于提供关于顺铂在纳米管生物偶联物上负载的额外信息。

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.