Gregg R. Dieckmann

Are carbon nanotubes a natural solution? Applications in biology and medicine.

Carbon nanotubes and materials based on carbon nanotubes have many perceived applications in the field of biomedicine. Several highly promising examples have been highlighted in the literature, ranging from their use as growth substrates or tissue scaffolds to acting as intracellular transporters for various therapeutic and diagnostic agents. In addition, carbon nanotubes have a strong optical absorption in the near-infrared region (in which tissue is transparent), which enables their use for biological imaging applications and photothermal ablation of tumors. Although these advances are potentially game-changing, excitement must be tempered somewhat as several bottlenecks exist. Carbon nanotube-based technologies ultimately have to compete with and out-perform existing technologies in terms of performance and price. Moreover, issues have been highlighted relating to toxicity, which presents an obstacle for the transition from preclinical to clinical use. Although many studies have suggested that well-functionalized carbon nanotubes appear to be safe to the treated animals, mainly rodents, long-term toxicity issues remains to be elucidated. In this report, we systematically highlight some of the most promising biomedical application areas of carbon nanotubes and review the interaction of carbon nanotubes with cultured cells and living organisms with a particular focus on in vivo biodistribution and potential adverse health effects. To conclude, future challenges and prospects of carbon nanotubes for biomedical applications will be addressed.

Single-walled carbon nanotube interactions with HeLa cells

This work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs) dispersed in cell culture media supplemented with serum. First, the as-received CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma – mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time- and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOX™ Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

Amphiphilic helical peptide enhances the uptake of single-walled carbon nanotubes by living cells

The success of many projected applications of carbon nano-tubes (CNTs) to living cells, such as intracellular sensors and nanovectors, will depend on how many CNTs are taken up by cells. Here we report the enhanced uptake by HeLa cells of single-walled CNTs coated with a designed peptide termed nano-1. Atomic force microscopy showed that the dispersions were composed of individual and small bundles of nano-1 CNTs with 0.7- to 32-nm diameters and 100- to 400-nm lengths. Spectroscopic characterizations revealed that nano-1 disperses CNTs in a non-covalent fashion that preserves CNT optical properties. Elemental analyses indicated that our sample preparation protocol involving sonication and centrifugation effectively eliminated metal impurities associated with CNT manufacturing processes. We further showed that the purified CNT dispersions are taken up by HeLa cells in a time- and temperature-dependent fashion, and that they do not affect the HeLa cell growth rate, evidence that the CNTs inside cells are not toxic under these conditions. Finally, we discovered that ~6-fold more CNTs are taken up by cells in the presence of nano-1 compared with medium containing serum but no peptide. The fact that coating CNTs with a peptide enhances uptake offers a strategy for improving the performance of applications that require CNTs to be inside cells.

Molecular dynamics study of a nanotube-binding amphiphilic helical peptide at different water/hydrophobic interfaces.

Many potential applications of single-walled carbon nanotubes (SWNTs) require that they be isolated from one another. This may be accomplished through covalent or noncovalent SWNT functionalization. The noncovalent approach preserves the intrinsic electrical, optical, and mechanical properties of SWNTs and can be achieved by dispersing SWNTs in aqueous solution using surfactants, polymers, or biomacromolecules like DNA or polypeptides. The designed amphiphilic helical peptide nano-1, which contains hydrophobic valine and aromatic phenylalanine residues for interaction with SWNTs and glutamic acid and lysine residues for water solubility, has been shown to debundle and disperse SWNTs, although the details of the peptide-SWNT interactions await elucidation. Here we use fully atomistic molecular dynamics simulations to investigate the nano-1 peptide at three different water/hydrophobic interfaces: water/oil, water/graphite, and water/SWNT. The amphiphilic nature of the peptide is characterized by its secondary structure, peptide-water hydrogen bonding, and peptide-hydrophobic surface van der Waals energy. We show that nano-1 has reduced amphiphilic character at the water/oil interface because the peptide helix penetrates into the hydrophobic phase. The peptide alpha-helix cannot match its hydrophobic face to the rigid planar graphite surface without partially unfolding. In contrast, nano-1 can curve on the SWNT surface in an alpha-helical conformation to simultaneously maximize its hydrophobic contacts with the SWNT and its hydrogen bonds with water. The molecular insight into the peptide conformation at the various hydrophobic surfaces provides guidelines for future peptide design.

Molecular imprinting of mesoporous SBA-15 with chiral ruthenium complexes

Abstract A novel strategy for imprinting mesoporous SBA-15 using chiral ruthenium complexes is reported. This imprinting strategy combines the triblock copolymer (PEO) 20 (PPO) 70 (PEO) 20 template and the chiral dichlorotris(1,10-phenanthroline)ruthenium(II) complex. A chiral PEO helix is formed by interaction of the chiral ruthenium complex with the block copolymer during the templated synthesis of the mesoporous SBA-15 molecular sieves. Upon removal of the chiral ruthenium complex, a stereospecific cavity is created. Preliminary results indicate that stereoselective absorption of either Δ or Λ -Ru(phen) 3 2+ isomers from a racemic mixture can be achieved depending on the chirality of the PEO chain.

A carbon nanotube-based Raman-imaging immunoassay for evaluating tumor targeting ligands

Herein, we describe a versatile immunoassay that uses biotinylated single-walled carbon nanotubes (SWNTs) as a Raman label, avidin-biotin chemistry to link targeting ligands to the label, and confocal Raman microscopy to image whole cells. Using a breast tumor cell model, we demonstrate the usefulness of the method to assess membrane receptor/ligand systems by evaluating a monoclonal antibody, Her-66, known to target the Her2 receptors that are overexpressed on these cells. We present two-dimensional Raman images of the cellular distribution of the SWNT labels corresponding to the distribution of the Her2 receptors in different focal planes through the cell with validation of the method using immunofluorescence microscopy, demonstrating that the Her-66-SWNT complexes were targeted to Her2 cell receptors.

Adsorption of Naphthalene and Pyrene Containing Surfactant Peptides onto Single- Walled Carbon Nanotubes: A Microscopy, Spectroscopy, and Theoretical Study

We examined the interaction between polycyclic aromatic hydrocarbons (PAHs) and single-walled carbon nanotubes (SWCNTs) using both experiments and molecular dynamics (MD) simulations. PAH molecules have a hexagonal arrangement of sp-hybridized carbon atoms, the same structural motif shared by carbon nanomaterials such as graphene and carbon nanotubes. Therefore, PAHs form π-π stacking interactions with other graphitic surfaces and can be used as dispersal agents for SWCNTs [1].

Microscopy, Fluorescence, and Confocal Raman Imaging of Biotinylated Single-Walled Carbon Nanotubes Bound to Breast Tumor Cells

Surface functionalization of carbon nanotubes is a strategy for the synthesis of SWNT-targeting moiety constructs and allows these hybrid nanotube-conjugates to be useful in various biomedical applications [1-2]. In this work, carboxylated single-walled carbon nanotubes (C-SWNTs), chemically modified by nitric acid reflux, were covalently coupled to a tether of (+)-Biotinyl-3,6,9trioxaundecanediamine (biotin-LC-PEO-amine) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The SWNT-(biotin-LC-PEO) amine constructs (B-SWNTs) were characterized by microscopic and spectroscopic methods. A sensitive and versatile sandwich immunoassay design was then developed that takes advantage of the specific interaction between Her2 receptors on breast tumor cells (BT-474 cell line) and a monoclonal antibody (Her-66) specific for the receptor. A biotinylated secondary antibody that recognizes the primary antibody was used to tag a NeutrAvidin-FITC marker via the strong binding affinity between biotin and avidin. BSWNTs were then targeted specifically to bind available sites on the NeutrAvidin-FITC. The surface distribution of receptors on BT-474 cells, indirectly marked by NeutrAvidin-FITC bound to the antibody, was studied by immunofluorescence microscopy at 15 and 37 C. Confocal Raman imaging was used to probe the localization of SWNT binding to the tumor cells.