Are Carbon Nanotubes Toxic?

Carbon Nanotube (CNT) is an extremely important and well-known nanomaterial widely used in drug delivery.¹ Although it is largely non-toxic, prolonged exposure at higher concentrations could lead to inflammation, generation of oxidative stress, cytotoxicity, and genotoxicity.² This article explores the factors contributing to CNT toxicity and discusses preventive measures.

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What are Carbon Nanotubes?

CNTs are cylindrical nanostructures that are composed of carbon atoms. These nanomaterials were first discovered in 1991 by Sumio Iijima. CNTs are classified into two groups: single-wall Carbon Nanotube (SWCNT) and the multi-wall carbon nanotubes (MWCNTs).³

SWCNTs comprise a single graphite layer, while MWCNTs contain multilayer concentric graphite. The exceptional tensile strength, biocompatibility, and electrical and thermal properties of CNTs are attributed to their unique nanostructure.⁴ These properties also broaden their applications in medicine, nanosensors, hydrogen storage, and field emission devices.

CNTs are commonly used in nanomedicine, particularly for targeted drug delivery. In this context, these nanomaterials are introduced to humans through various routes, including oral, inhalation, intravenous (IV) injection, transdermal, intraperitoneal, and subcutaneous injection.⁵ 

In living cells, CNTs typically enter through passive diffusion or energy-dependent endocytosis.⁶ They get distributed in different parts of the body and may eventually remain, translocate, or be excreted from the body. In vivo experiments have indicated the accumulation of CNTs in the spleen, kidneys, and lungs.²

Toxicological Considerations of CNTs

Exposure to pure CNTs has been shown to reduce cell proliferation rate. It also induces apoptosis, cell cycle arrest, and necrosis.⁷ Mechanistically, pristine CNTs interact with proteins and modify their structure, ultimately leading to cell death.

Several studies have shown that oral, IV injection, and dermal administration of CNTs can lead to mild inflammation in humans. Compared to the aforementioned routes, CNT exposure through inhalation results in severe inflammation.²

Since CNTs easily translocate to different parts of the body, it is important to understand their effects on different organs.

When CNTs enter cells through the cellular lipid bilayer membrane, they induce oxidative stress that may result in inflammation and cytotoxicity.⁸ Nuclear factor-Kappa B and protein kinases are two key signaling factors that regulate the release of cytokines in response to CNT-induced oxidative stress. The high levels of free radicals generated by oxidative stress can damage proteins, lipids, and DNA within cells.

As CNTs increase oxidative stress levels within a cell, they enhance reactive oxygen species (ROS) levels, contributing to cytotoxicity. An increased ROS level also leads to oxidation of amino acids, inactivation of enzymes, apoptosis, and disruption of genetic materials.⁹

Living cells exposed to SWCNTs at a concentration of 200 μg/mL have been shown to exhibit a significant increase in ROS levels.² An increased inflammatory response triggered through CNT exposure has also been associated with the generation of cell toxicity. A continual accumulation of CNTs in the lungs causes black spots. 

An in vivo experiment that exposed mice to MWCNTs induced inflammation and granuloma formation in the lung airways.¹⁰ The accumulation of SWCNTs in the liver induced inflammation, cell proliferation, and elevated enzyme production, particularly aspartate aminotransferase and bilirubinemia, leading to hepatotoxicity. 

Factors Linked to CNT’s Toxicity

Numerous key factors affect the toxicity of CNTs.

Purity

During CNT synthesis, metals such as iron, nickel, cobalt, and molybdenum are used as catalysts. Metal impurities have been identified as a critical factor in determining CNT toxicity. CNTs containing metallic impurities can induce mitochondrial destruction and oxidative stress. To address this, several CNT purification methods have been designed, such as ultra-sonication.⁷

Shape 

The shape of nanomaterials significantly contributes to their toxicity. In general, rod-shaped nanostructures are more toxic than spherical ones. Nanomaterials with larger aspect ratios are also more toxic than smaller ones.¹¹

For instance, an in vivo experiment revealed that when SWCNT and MWCNT were injected into a rat’s artery, vascular thrombosis and platelet aggregation occurred. In contrast, the injection of fullerenes, a spherical carbon nanomaterial, did not result in such outcomes.

Size 

The length and diameter of CNTs also determine toxicity. When the length of CNT exceeds the length of the macrophage, ‘frustrated’ phagocytosis occurs. This prevents the complete removal of CNTs from the system and leads to a release of inflammatory factors. It is noteworthy that smaller-sized CNTs have shown no toxicity.

Functionalization

Functionalizing CNTs before using them in a biological environment is important. This process can significantly reduce their toxicity and improve biocompatibility and dispersibility. 

Novel Strategies to Mitigate CNT Toxicity

Several strategies have been formulated to mitigate the inherent toxicity of CNT. Research involving rodents has shown that functionalizing MWCNTs can eliminate their toxicity. A common functionalization method involves coating the surface of CNTs with polyethylene glycol (PEG).

Several in vivo studies have documented that CNT functionalized with PEG improved pharmacokinetic behavior and reduced toxicity and reticuloendothelial system (RES) capture.⁷ It also improved prolonged CNT’s blood circulation half-life. Unlike pristine CNT, PEG-coated CNT is excreted through urine and feces. Appropriate functionalization can significantly reduce CNT toxicity, even at higher doses. The length of MWCNTs can also be modified to reduce toxicity. Chemical modification of the CNT surface also reduces toxicity.

A recently developed curcumin-coated lysine functionalized MWCNTs exhibited decreased levels of inflammatory molecules, ROS production, and upregulation of the antioxidant enzyme catalase. The curcumin coating also promoted recovery of mitochondrial membrane potential in the MWCNTs exposed cells.¹²

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References and Further Reading

1. Zare, H., et al. (2021). Carbon Nanotubes: Smart Drug/Gene Delivery Carriers. Int J Nanomedicine. doi.org/10.2147/IJN.S338281
2. Mohanta, D., Patnaik, S., Sood, S., Das N. (2019). Carbon nanotubes: Evaluation of toxicity at biointerfaces. J Pharm Anal. doi.org/10.1016/j.jpha.2019.04.003
3. Aqel, A., et al. (2012). Carbon nanotubes, science and technology part (I) structure, synthesis, and characterisation. Arab J Chem. doi.org/10.1016/j.arabjc.2010.08.022
4. Ferrier, DC., Honeychurch KC. (2021). Carbon Nanotube (CNT)-Based Biosensors. Biosensors (Basel). doi.org/10.3390/bios11120486
5. Zhang, W., Zhang, Z., Zhang, Y. (2011). The application of carbon nanotubes in target drug delivery systems for cancer therapies. Nanoscale Res Lett. doi.org/10.1186/1556-276X-6-555
6. Yaron, PN., et al. Single-wall carbon nanotubes enter cells by endocytosis and not membrane penetration. J Nanobiotechnology. doi.org/10.1186/1477-3155-9-45
7. Madani, SY., Mandel, A., Seifalian, AM. (2013). A concise review of carbon nanotube’s toxicology. Nano Rev. doi.org/10.3402/nano.v4i0.21521
8. Gao, S., Xu, B., Sun, J., Zhang, Z. (2024). Nanotechnological advances in cancer: therapy a comprehensive review of carbon nanotube applications. Front Bioeng Biotechnol. doi.org/10.3389/fbioe.2024.1351787
9. Juan, CA., et al. (2021). The Chemistry of Reactive Oxygen Species (ROS) Revisited: Outlining Their Role in Biological Macromolecules (DNA, Lipids and Proteins) and Induced Pathologies. International Journal of Molecular Sciences. doi.org/10.3390/ijms22094642
10. Barna, BP., Malur, A., Thomassen, MJ. (2021). Studies in a Murine Granuloma Model of Instilled Carbon Nanotubes: Relevance to Sarcoidosis. Int J Mol Sci. doi.org/10.3390/ijms22073705
11. Huang, YW., Cambre, M., Lee, HJ. (2017). The Toxicity of Nanoparticles Depends on Multiple Molecular and Physicochemical Mechanisms. Int J Mol Sci. doi.org/10.3390/ijms18122702
12. Rele, S, et al. (2024). Curcumin coating: a novel solution to mitigate inherent carbon nanotube toxicity. J Mater Sci Mater Med. doi.org/10.1007/s10856-024-06789-9

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