Internalization Mechanism of carbon nanotube

Internalization mechanism of carbon nanotube and their distribution within Mammalian Cell Line

Several applications of CNTs in the field of biomedical such as drug delivery, gene delivery, biosensor and intracellular transporters are due to the important features exhibited by this novel material which shows high propensity to traverse cell membrane and highly efficient molecular deliver into mammalian cells. Small molecules, large proteins and amino acids previously demonstrated minimal cellular penetration can be easily increase by using carbon nanotubes (Klumpp et al., 2006).

Studies conducted by few researchers, for example Pantarotto et al. (2004) and Kam et al. (2005) revealed that functionalized CNTs are capable in traversing cell membrane with higher efficiency rather than pure CNTs. Investigation by Cui et al. (2005) and Tian et al. (2006) found that pure CNTs were very difficult to enter HEK 293 cells, human fibroblast cells and stem cells. These evidences revealed that modified CNTs surface by simply attaching biomolecules such as DNA, protein, peptide, oligonucleotide, nucleic acid, and siRNA, demonstrated higher efficiency in entering almost all cells ranging from normal primary cultured, tumor cells to stem cells rather than non modified CNTs which enter cells by very low efficient means.

Currently, by which pathways CNTs entering cells, their localization and fate after internalization are becoming the central question and controversial topic. Different routes of the uptake mechanisms for CNTs entering cells have been proposed by researchers, but until now there is no agreement between one another. First study regarding the uptake mechanism of CNTs has been carried out by Pantarotto and his colleagues (Pantarotto et al., 2004). They observed the uptake of water soluble, amino functionalized SWNTs conjugated fluorescent dye, FITC and peptide in human and murine fibroblast (3T6 and 3T3), keratinocytes and HeLa cell lines. Both conjugates were internalize by those cell lines, however their site of accumulation tend to differ. Peptide-SWNTs were mainly found to accumulate in the nucleus while directly labeled SWNTs localized in the cytoplasm. In their investigation, they observed that the uptakes were not influenced by the changes in temperature (37 0C or 40C) and in the present of endocytosis inhibitors, sodium azide. As a result, they suggested that CNTs uptake mechanism to be energy independent non endocytotic. Mechanism of nanotube uptake indicated that it occurs via insertion or penetration through the plasma membrane with the hypothesized that functionalized carbon nanotubes have cylindrical shape and high aspect ratio which allows them to pierce plasma membrane.

Inspired by recent experimental investigations that CNTs can enter mammalian cells, Gao et al. (2008) demonstrated the interaction of carbon nanotube and cells based on coarse grain molecular dynamics and theoretical studies in order to understand the mechanism of nanotube entering cell membrane, specifically interaction mechanisms between CNTs and lipid bilayer. Their results indicated that the interaction is dominated mainly by weak van der Waals and hydrophobic forces. Small radius nanotubes can directly penetrate through cell membrane while the larger tubes likely to use wrapping mechanism.

Kam et al. (2006) presented a review regarding various potential biomedical application of carbon nanotube in particular as a cellular transporter and delivery system for biological molecules such as DNA or siRNA (Kam et al., 2006). The mechanism of internalization for SWNTs entering mammalian cells claimed to be clathrin-mediated endocytosis due to the systematic study of cellular uptake mechanism and pathway for carbon nanotube (Kam et al., 2005). Uptake by cell found to be hindered at lower temperature (40C) and in the absence of ATP, where cells pretreated with endocytosis inhibitor, sodium azide. Authors found that different cell types such as HL 60 cells, Jurkat, Chinese Hamster Ovary (CHO) and 3T3 fibroblasts also showed the temperature dependent uptake. Simultaneously, SWNTs internalized inside mammalian cells show signs of colocalization with red endosome marker, FM 4-64. These evidences help authors to declare that carbon nanotubes entering cells via energy dependent endocytosis process. In their studies, Kam and co-workers (Kam et al., 2004, 2005) used protein-oxidized SWNTs functionalized covalently (Kam et al., 2004) and non-covalently (Kam et al., 2005, 2006) where the length and diameter of SWNTs in the range of hundreds of nm and 1-5 nm respectively. The length of nanotubes (~ 100 nm) used in authors' study match up the size of particles that can enter cells via clathrin coated endocytosis.

Another proposed mechanism for nanotubes entering cells is phagocytosis. Cherukuri et al. (2004) incubated different concentration of SWNTs with mouse peritoneal macrophages cells in growth media containing surfactant, Pluronic. Due to the type of cell and the average length of nanotubes used (1 µm), the mechanism for nanotubes entering cells associated with phagocytosis. This type of uptake restricted only to specialized cell like monocytes and macrophages and the most important is the size of particles which can be taken up by the cells through this mechanism, where it is in the range of 0.1-10 µm. In this experiment, nanotubes tend to accumulate in intracellular regions apparently in the small phagosomes in the cytoplasm. Authors suggested that nanotubes were rapidly engulfed through phagocytosis in temperature-dependent manner.

In recent study, Cheng and co-workers (Cheng et al., 2008) investigated the intracellular distribution of FITC-labeled, PEGylated SWNTs in several cell lines and they reported the accumulation of carbon nanotubes in the nucleus, mainly in the nucleolus of HeLa, U2OS and HT1080 cells. Simultaneously, no accumulation was detected in the nucleolus of MEF, C33A and HEK293 cells. They proposed that CNTs remained in the endosome for shorter time rather than Dextran or both CNTs and Dextran entering cells via different routes.

In contrast to studies on how nanotube entering cells, Strano et al. (2008) monitored and assessed the exocytosis process of SWNTs and found that the rate is closely comparable to the rate of endocytosis in NIH-3T3 cells with negligible temporal offset.

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