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Abstract

  1. Top of page
  2. Abstract
  3. Overview of Asbestos-Induced Carcinogenesis
  4. Translocation and Biopersistence of Fibers
  5. Mesothelial and Epithelial Cell Injury Induced by Fibrous Materials
  6. Factors Involved in Carbon Nanotube and Asbestos Fiber Uptake by Non-Phagocytic Cells
  7. Asbestos Fibers are Endocytosed by Mesothelial and Epithelial Cells
  8. Cellular Uptake of Carbon Nanotubes Varies
  9. Size and Surface Characteristics of Carbon Nanotubes Determine Uptake Pathways
  10. Carbon Nanotubes and Asbestos Fibers Activate Macrophages
  11. Concluding Remarks
  12. Acknowledgments
  13. Disclosure Statement
  14. References

The emergence of nanotechnology represents an important milestone, as it opens the way to a broad spectrum of applications for nanomaterials in the fields of engineering, industry and medicine. One example of nanomaterials that have the potential for widespread use is carbon nanotubes, which have a tubular structure made of graphene sheets. However, there have been concerns that they may pose a potential health risk due to their similarities to asbestos, namely their high biopersistence and needle-like structure. We recently found that despite these similarities, carbon nanotubes and asbestos differ in certain aspects, such as their mechanism of entry into mesothelial cells. In the study, we showed that non-functionalized, multi-walled carbon nanotubes enter mesothelial cells by directly piercing through the cell membrane in a diameter- and rigidity-dependent manner, whereas asbestos mainly enters these cells through the process of endocytosis, which is independent of fiber diameter. In this review, we discuss the key differences, as well as similarities, between asbestos fibers and carbon nanotubes. We also summarize previous reports regarding the mechanism of carbon nanotube entry into non-phagocytic cells. As the entry of fibers into mesothelial cells is a crucial step in mesothelial carcinogenesis, we believe that a comprehensive study on the differences by which carbon nanotubes and asbestos fibers enter into non-phagocytic cells will provide important clues for the safer manufacture of carbon nanotubes through strict regulation on fiber characteristics, such as diameter, surface properties, length and rigidity. (Cancer Sci, doi: 10.1111/j.1349-7006.2012.02326.x, 2012)

Nanotechnology is an emerging technology with the potential to make our daily lives better, as long as we know how it works.[1] Asbestos, a natural fibrous silicate mineral, was once hailed as a miraculous stone and used throughout the world during the 20th century; however, it is now referred to as a time bomb due to its carcinogenicity through inhalation.[2] The mechanism of asbestos-induced carcinogenesis is yet to be fully unraveled, but it is currently known that asbestos fibers' physical characteristics, especially high biopersistence and needle-like structure, are among the crucial factors responsible for causing cancer.[3, 4] Carbon nanotubes are a nanomaterial that was recently discovered, manufactured, and commercially distributed. These tube-shaped, carbon nanomaterials have raised social concerns due to their strong resemblance to asbestos fibers, especially in regard to the two features mentioned above.

The toxicological similarity of carbon nanotubes and asbestos fibers was reported by Poland et al.[5] They reported that long fibers, whether they are carbon nanotubes or asbestos fibers, evade complete phagocytosis by macrophages and cause inflammation. This failure in phagocytosis, termed frustrated phagocytosis, has gained attention as a potential mechanism to explain the inflammogenicity and carcinogenicity of fibers.[6-8]

However, despite their similarities in certain aspects, carbon nanotubes and asbestos fibers vary in many physicochemical features, including constituent element, weight, and surface property. Therefore, we believe that these two distinct types of fibers do not necessarily exert toxicological effects through the same mechanism. One key difference that distinguishes these two fibers might be the mechanism of fiber entry into mesothelial cells. Considering that fiber entry into mesothelial cells is a key step in carcinogenesis, the difference between nanotubes and asbestos may provide a clue to aid in the development of safer carbon nanotubes. Moreover, the elucidation of the mechanism by which nanotubes enter into cells is of great use in both toxicology and medical applications.[9]

In this review, to establish a difference between the toxicological paradigms of carbon nanotubes and asbestos fibers, we summarize the current understanding of the differences and similarities between these two fibrous materials, with an emphasis on the entry mechanism of carbon nanotubes and asbestos fibers into non-phagocytic cells.

Overview of Asbestos-Induced Carcinogenesis

  1. Top of page
  2. Abstract
  3. Overview of Asbestos-Induced Carcinogenesis
  4. Translocation and Biopersistence of Fibers
  5. Mesothelial and Epithelial Cell Injury Induced by Fibrous Materials
  6. Factors Involved in Carbon Nanotube and Asbestos Fiber Uptake by Non-Phagocytic Cells
  7. Asbestos Fibers are Endocytosed by Mesothelial and Epithelial Cells
  8. Cellular Uptake of Carbon Nanotubes Varies
  9. Size and Surface Characteristics of Carbon Nanotubes Determine Uptake Pathways
  10. Carbon Nanotubes and Asbestos Fibers Activate Macrophages
  11. Concluding Remarks
  12. Acknowledgments
  13. Disclosure Statement
  14. References

Before describing the similarities and differences between carbon nanotubes and asbestos fibers, we briefly outline the mechanism of asbestos-induced carcinogenesis. Asbestos fibers are inhaled and eventually reach the pulmonary alveoli. During this process, asbestos fibers come into contact with epithelial cells in the trachea, bronchus and alveoli, as well as alveolar macrophages, leading to epithelial cell injury and macrophage activation. Small particles are well phagocytosed by macrophages and are excreted from the respiratory system via the lymphatic system. However, other long or large fibers that evade phagocytosis are not cleared and stay inside the lung for a long period of time, leading to chronic inflammation. Fibers that remain in the lung can physically penetrate through alveolar epithelial cells and visceral mesothelial cells and reach the parietal mesothelial cells due to negative pressure in the pleural cavity. Donaldson et al. hypothesized that long fibers (length > ~15 μm) tend to be trapped around the parietal stomata and the entryways to lymphatic vessels, as they cannot flow into the small openings like short fibers. Furthermore, they argued that mesothelial cell injury and macrophage activation occur at these sites of fiber accumulation, which then lead to the formation of mesothelioma.[3]

To date, we have four main criteria to assess toxicological and carcinogenic features of fibers: (i) translocation of fibers, such as from trachea to alveoli and from alveoli to parietal pleura; (ii) biopersistence, which determines the longevity of fibers inside organisms; (iii) mesothelial or epithelial cell injury caused by fibers; and (iv) activation of macrophages that phagocytosed the fibers. We will briefly review the translocation and biopersistence of fibers. Thereafter, we will provide an in-depth discussion of the differences between carbon nanotubes and asbestos fibers in the mechanism of mesothelial or epithelial cell injury, with an emphasis on the cellular entry mechanism of these fibers. We will also compare the effects of carbon nanotubes and asbestos fibers on macrophage activation.

Translocation and Biopersistence of Fibers

  1. Top of page
  2. Abstract
  3. Overview of Asbestos-Induced Carcinogenesis
  4. Translocation and Biopersistence of Fibers
  5. Mesothelial and Epithelial Cell Injury Induced by Fibrous Materials
  6. Factors Involved in Carbon Nanotube and Asbestos Fiber Uptake by Non-Phagocytic Cells
  7. Asbestos Fibers are Endocytosed by Mesothelial and Epithelial Cells
  8. Cellular Uptake of Carbon Nanotubes Varies
  9. Size and Surface Characteristics of Carbon Nanotubes Determine Uptake Pathways
  10. Carbon Nanotubes and Asbestos Fibers Activate Macrophages
  11. Concluding Remarks
  12. Acknowledgments
  13. Disclosure Statement
  14. References

Based on clinical observations, malignant mesothelioma develops most frequently at the parietal pleura, which unlike the visceral pleura, is not directly adjacent to the lung parenchyma. Appearance at the parietal pleura is reflected in the clinical stage. Such findings suggest that asbestos fibers somehow migrate through the lung parenchyma and reach the pleural cavity, where they come into contact with the parietal pleura. The exact mechanism by which fibers finally reach the parietal pleural has yet to be confirmed; however, Miserocchi et al.[10] suggested that fibers in alveolar space travel physically through the paracellular pathway to reach the interstitial space between the lung parenchyma and visceral pleura. Then, the fibers are able to translocate to almost all organs via fluid flow, such as lymph and blood. Finally, some fibers get stuck in the parietal pleura, especially around the openings of the lymphatic stomata. An alternate hypothesis is that fibers directly translocate through the lung parenchyma and visceral pleura to reach the parietal mesothelium.[11] Although the high physical resistance of the visceral pleura is difficult for any fiber to migrate through, it can still take place during chronic inflammation. During inflammation, the interstitial space exerts higher pressure, making it is easier for fibers to pass through the visceral pleura.[10] Regardless of whether the fibers travel through the vascular system or directly translocate, they will be cleared by the lymphatic stomata when they reach the pleural cavity, and this pathway may partially explain why longer fibers (>8 μm), which are more likely to be trapped around the stomata compared to shorter fibers, are more carcinogenic in vivo.[3, 12]

The translocation of inhaled carbon nanotubes to subpleural tissue has been reported.[13] The nanotubes used were suspended in phosphate-buffered saline containing pluronic F-68. As will be discussed later, the surface characteristics and size of nanotubes greatly affect their behavior. For example, carbon nanotubes that were covalently bonded with amino groups were easily distributed to each organ and ultimately excreted in the urine.[14] This type of modification is called functionalization and is often performed to relieve the strong hydrophobicity of carbon nanotubes. The type of functional groups present on the surface of carbon nanotubes controls their hydrophilic/hydrophobic properties, which play important roles in the translocation, distribution and excretion of carbon nanotubes inside organisms. While this variable property of carbon nanotubes and its influence on cells is of interest, the biological behavior and effects of functionalized nanotubes should be carefully differentiated from those of pristine, or non-functionalized nanotubes, which are the ones that are industrially produced, commercially distributed and of toxicological concerns. Despite the fact that functionalized nanotubes show little toxicity, it is currently unrealistic to functionalize high amounts of pristine nanotubes. Therefore, it is essential to carefully consider how pristine nanotubes would behave inside organisms for the toxicological assessment of carbon nanotubes.

Biopersistence is defined as the durability of materials inside organisms and is known to affect fiber translocation. Highly biopersistent fibers contribute to sustained inflammation and cell injury followed by the development of cancer.[15] Clearance of fibers is affected by the dimension of the fiber, as long fibers that fail to be phagocytosed by macrophages are not carried and excreted.[3] Furthermore, as mentioned above, long fibers are likely to be trapped at lymphatic stomata, leading to a failure of fiber clearance.[12] In addition to the fiber dimension, the chemical composition of asbestos and carbon nanotubes renders them chemically stable, which enhances biopersistence because biodegradable fibers would be diminished following macrophage digestion. Mutlu et al.[16] showed that carbon nanotubes that were well dispersed in pluronic solution were gradually cleared from the mouse lung. However, the translocation and biopersistence of “pristine” carbon nanotubes that are not coated by modifying materials should be investigated to gain further understanding of the toxicological property of carbon nanotubes.

Mesothelial and Epithelial Cell Injury Induced by Fibrous Materials

  1. Top of page
  2. Abstract
  3. Overview of Asbestos-Induced Carcinogenesis
  4. Translocation and Biopersistence of Fibers
  5. Mesothelial and Epithelial Cell Injury Induced by Fibrous Materials
  6. Factors Involved in Carbon Nanotube and Asbestos Fiber Uptake by Non-Phagocytic Cells
  7. Asbestos Fibers are Endocytosed by Mesothelial and Epithelial Cells
  8. Cellular Uptake of Carbon Nanotubes Varies
  9. Size and Surface Characteristics of Carbon Nanotubes Determine Uptake Pathways
  10. Carbon Nanotubes and Asbestos Fibers Activate Macrophages
  11. Concluding Remarks
  12. Acknowledgments
  13. Disclosure Statement
  14. References

The carcinogenic feature of asbestos fibers and carbon nanotubes has gained a great deal of attention from both scientists and the public. Fibrous materials have the potential to transform normal cells, constituting a well-organized tissue, into cancer cells by causing chromosomal aberrations and/or gene mutations that lead to invasion and destruction of the surrounding tissue. The target cells in fibrous material-induced carcinogenesis are mesothelial cells or epithelial cells, but not macrophages. Therefore, the mechanism by which the DNA of mesothelial/epithelial cells is damaged has been of great interest. Currently, there are four key phenomena by which fibrous materials are postulated to cause DNA damage: (i) free radical generation on the surface; (ii) physical interaction between DNA and fibrous materials; (iii) molecule adsorption on the material surface; and (iv) chronic inflammation, as previously described.[4] All of these mechanisms might take place simultaneously during fibrous material-induced carcinogenesis, but the relative contribution of each may differ between carbon nanotubes and asbestos fibers. For example, carbon nanotubes piercing through the cellular and nuclear membrane might be more likely to directly associate with chromosomes than endocytosed asbestos fibers because penetrating carbon nanotubes are not covered by a membrane structure that would separate materials from the nuclear components (Fig. 1), as discussed previously.[17]

image

Figure 1. Distinct uptake mechanism of asbestos fibers and carbon nanotubes by mesothelial cells. Chrysotile asbestos fibers (red arrows) are taken up by a mesothelial cell. Surrounding membranous structure around asbestos (blue arrowheads) indicates that the asbestos is endocytosed. On the other hand, a carbon nanotube (red arrows; diameter = ~50 nm) directly pierces the cellular and nuclear membrane. The human peritoneal mesothelial cell line was established as previously described.[17] The cells were fixed after a 3 h-incubation with indicated fibers. To increase the resolution, ultrathin sections were prepared at 80 nm thickness for asbestos. Considering the fact that carbon nanotubes are too rigid for cutting with diamond-knife, the sections were prepared at 500 nm thickness for nanotube.

Download figure to PowerPoint

Non-phagocytic cells, such as epithelial and mesothelial cells, form a continuous cell layer that acts as a barrier to cover and protect the underlying structure. Following exposure to fibrous materials, some of the cells undergo apoptosis or programmed necrosis, while others are able to evade such cell death mechanisms.[18-20] The remaining live cells that hold these fibers inside are the most likely to become cancer cells after fibrous material-induced DNA damage. As a consequence of DNA damage in mesothelial cells, homozygous deletion of the Cdkn2a/2b tumor suppressor genes is frequently observed, not only in asbestos-induced mesothelioma[21, 22] but also in carbon nanotube-induced mesothelioma.[17] We previously showed that mesothelioma induced by iron saccharate also displayed homozygous deletion of Cdkn2a/2b,[23] suggesting that the loss of these genes might be a major event in mesothelial carcinogenesis.

Factors Involved in Carbon Nanotube and Asbestos Fiber Uptake by Non-Phagocytic Cells

  1. Top of page
  2. Abstract
  3. Overview of Asbestos-Induced Carcinogenesis
  4. Translocation and Biopersistence of Fibers
  5. Mesothelial and Epithelial Cell Injury Induced by Fibrous Materials
  6. Factors Involved in Carbon Nanotube and Asbestos Fiber Uptake by Non-Phagocytic Cells
  7. Asbestos Fibers are Endocytosed by Mesothelial and Epithelial Cells
  8. Cellular Uptake of Carbon Nanotubes Varies
  9. Size and Surface Characteristics of Carbon Nanotubes Determine Uptake Pathways
  10. Carbon Nanotubes and Asbestos Fibers Activate Macrophages
  11. Concluding Remarks
  12. Acknowledgments
  13. Disclosure Statement
  14. References

Although the exact mechanism of DNA damage is yet to be elucidated, uptake of fibrous material by non-phagocytic cells appears an essential step in carcinogenesis. Furthermore, we believe that the key factor needed to differentiate carbon nanotube toxicology from that of asbestos fiber is the mechanism by which carbon nanotubes and asbestos fibers enter non-phagocytic cells. As we have recently reported, carbon nanotubes directly pierce through the cell membrane to enter mesothelial cells in a diameter- and rigidity-dependent manner, whereas asbestos fibers are actively endocytosed by these cells regardless of their diameter.[17] This study was the first direct evidence describing the toxicological difference between carbon nanotubes and asbestos fibers regarding the mechanism of the entry of these materials into mesothelial cells. In this review, we summarize the previous literature describing how non-phagocytic cells take up carbon nanotubes and asbestos fibers to refine and expand our understanding.

Many factors that are involved in the entry of fibrous materials into non-phagocytic cells can be roughly divided into two categories: material characteristics and cell condition. Fibrous materials vary largely from each other in terms of their length, diameter, constituents, surface area, volume and weight. In addition to such physical variations, carbon nanotubes often contain contaminant metals because they are chemically synthesized in the presence of metal catalysts, while asbestos fibers are mined from mountains. It is also of note that the methods used to prepare suspensions of fibrous materials and the assays used for their characterization differ from lab to lab, which could account for differing results. For in vitro studies, fibrous materials are often suspended in solutions that may contain salt, proteins, DNA, polymers or alcohol, which could influence the results. Although there is a vehicle control group in cell-based experiments, the possibility that these solvents might affect the interaction of cells with carbon nanotubes or asbestos fibers should be taken into account. Information regarding proteins or other molecules that are adsorbed onto fiber surfaces is described elsewhere and is known to affect fibrous material behavior and its effects on cells.[24, 25]

Cell type should also be carefully discussed in toxicology of asbestos fibers and carbon nanotubes. There are two primary cell types involved in this toxicology: non-phagocytic cells and phagocytic cells. The interaction of each cell type with fibrous materials is important in this toxicology, but the cellular behavior and its role in pathophysiological conditions are very different.

Asbestos Fibers are Endocytosed by Mesothelial and Epithelial Cells

  1. Top of page
  2. Abstract
  3. Overview of Asbestos-Induced Carcinogenesis
  4. Translocation and Biopersistence of Fibers
  5. Mesothelial and Epithelial Cell Injury Induced by Fibrous Materials
  6. Factors Involved in Carbon Nanotube and Asbestos Fiber Uptake by Non-Phagocytic Cells
  7. Asbestos Fibers are Endocytosed by Mesothelial and Epithelial Cells
  8. Cellular Uptake of Carbon Nanotubes Varies
  9. Size and Surface Characteristics of Carbon Nanotubes Determine Uptake Pathways
  10. Carbon Nanotubes and Asbestos Fibers Activate Macrophages
  11. Concluding Remarks
  12. Acknowledgments
  13. Disclosure Statement
  14. References

Because mesothelial and epithelial cells are not professional phagocytes, we should first address the question of whether asbestos fibers and carbon nanotubes enter into these cells when assessing the toxicity of these materials. The internalization of asbestos fibers into various non-phagocytic cells has been reported by many researchers (Fig. 1). Suzuki and Jaurand et al. beautifully demonstrated that alveolar epithelial cells and pleural mesothelial cells internalized chrysotile asbestos into the phagolysosome.[26, 27] Regarding the mechanism of the internalization, an interaction between the membrane proteins of the cells and proteins adsorbed onto the surface of asbestos fiber was investigated. Adsorption of vitronectin, a serum protein, onto the asbestos surface (chrysotile and crocidolite) was reported to enhance asbestos internalization by mesothelial and epithelial cells via the integrin alphaVbeta5 signaling pathways.[28-30] Following the internalization of asbestos fibers into mesothelial cells, we have observed in the previous study that the asbestos fibers were surrounded by the small GTPase, Rab5a, which is a marker of the early endosome and phagosome.[17, 31] Considering that the size of an endosome can be up to 1 μm,[32, 33] asbestos fibers that are larger than a few microns could be internalized by a phagocytosis-like mechanism rather than that of the endosome.

Cellular Uptake of Carbon Nanotubes Varies

  1. Top of page
  2. Abstract
  3. Overview of Asbestos-Induced Carcinogenesis
  4. Translocation and Biopersistence of Fibers
  5. Mesothelial and Epithelial Cell Injury Induced by Fibrous Materials
  6. Factors Involved in Carbon Nanotube and Asbestos Fiber Uptake by Non-Phagocytic Cells
  7. Asbestos Fibers are Endocytosed by Mesothelial and Epithelial Cells
  8. Cellular Uptake of Carbon Nanotubes Varies
  9. Size and Surface Characteristics of Carbon Nanotubes Determine Uptake Pathways
  10. Carbon Nanotubes and Asbestos Fibers Activate Macrophages
  11. Concluding Remarks
  12. Acknowledgments
  13. Disclosure Statement
  14. References

Carbon nanotubes are divided into two groups depending on their structure: single-walled carbon nanotubes (SWCNT) with a diameter of 1–3 nm and a length of 5–30 nm[34] and multi-walled carbon nanotubes (MWCNT) with a diameter of 3–200 nm and a length of tens of nanometers to microns.[35, 36] As discussed previously, the size of carbon nanotubes is an important determinant of how the carbon nanotubes enter non-phagocytic cells. Several reports have suggested that carbon nanotubes are able to enter non-phagocytic cells via endocytosis or membrane piercing; however, a reasonable explanation on the difference has yet to be established. To identify common features of carbon nanotubes that determine the mechanism of cell entry, we have summarized the current literature describing carbon nanotube entry into non-phagocytic cells, with a focus on length, diameter and surface functionalization of carbon nanotubes, cell type, entry mechanism and intracellular localization (Table 1).

Table 1. Analysis of cellular entry of carbon nanotubes in non-phagocytic cells in the literature
NanotubeLengthDiameterSurfaceCell typeToxicityMechanismIntracellular locationReferences
  1. BSA, bovine serum albumin; CMC, carboxymethyl cellulose; DPPC, dipalmytoilphosphatidylcholine; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FITC, fluorescein isothiocyanate; IL, interleukin; in ref, described in the reference(s); LDH, lactate dehydrogenase; MWCNT, multi-walled carbon nanotubes; ND, not described; PBS, phophate-buffered saline; PEG, polyethylene glycol; siRNA, small interfering RNA; SWCNT, single-walled carbon nanotube.

Endocytosis
SWCNT~100–1000 nm1–5 nmCovalently functionalized (oxidized) + streptavidinHL60 and JurkatLittle toxicityEndocytosisEndocytic vesicles [51]
SWCNTTens to hundreds of nm~1.5 nmCovalently functionalized (oxidized) + streptavidin, albumin, protein A or cytochrome cHeLa, NIH3T3, HL60 and JurkatLittle toxicityEndocytosisEndocytic vesicles [50]
SWCNTShort (145 nm) and long (1250 nm)0.7–1.3 nm in ref.Non-covalent binding with Pluronic F127HeLaNDEndocytosisPerinuclear localization [63]
MWCNT7000–10 000 nm80–130 nmNon-functionalized and suspended in PBS containing 0.1% gelatinMESO-1, BEAS-2B and IMR-32Toxicity dependent on cell typeEndocytosisPerinuclear localization without nuclear import [44]
SWCNTND1 nm in ref.Non-covalent binding with DNANIH3T3Little toxicityEndocytosis and exocytosisEndocytic vesicles including lysosomes [64]
SWCNT~150 nm~1.2 nmNon-covalent binding with DNA or phospholipid-conjugated folic acidHeLaSelective toxicity by coupling near infrared light and interaction of folic acid-nanotube and folate receptor-expressing cancer cellsFolate receptor-mediated endocytosisND [40]
SWCNT100–200 nm1–3 nmCovalently functionalized (oxidized) + chitosan, Alexa Fluor 488 and folic acidHepG2Little toxic up to 0.05 mg/mLFolate receptor-mediated endocytosisCytoplasm [42]
SWCNT100–300 nm1–5 nmCovalently functionalized (amino group, cisplatin and folic acid)KB, JAR and Ntera-2Selective toxicity by coupling cisplatin and interaction of folic acid-nanotube and folate receptor-expressing cancer cellsFolate receptor-mediated endocytosisEndosomes [39]
SWCNT50–100 nm1–3 nmCovalently functionalized (folic acid and Alexa Fluor 488)HepG2Selective toxicity by coupling near infrared light and interaction of folic acid-nanotube and folate receptor-expressing cancer cellsFolate receptor-mediated endocytosisCytoplasm [41]
SWCNT and MWCNTSWCNT (50–200 nm) and MWCNT (500–2000 nm)SWCNT (1–3 nm) and MWCNT (10–30 nm)Covalently functionalized (oxidized) + chitosan, Alexa Fluor 488 and folic acidHepG2Little toxic up to 0.01 mg/mL (as shown in Supporting information of reference 38)Both of endocytosis and piercing/diffusion happened. Addition of folic acid on MWCNTs enabled them to enter the cells via receptor-mediated endocytosisSize-dependent subcellular localization [38]
SWCNT300–1000 nm1–5 nmNon-covalent binding with PEG. EGF or folic acid was conjugated with PEGSKOV-3 and OVCA 433NDIntact but not fragmented PEG blocked non-specific uptake of nanotubes; conjugation of EGF or folic acid promoted specific cellular uptakeND [65]

MWCNT

(bamboo

structure)

3000–

10 000 nm

50–300 nmNon-functionalized and suspended in culture mediumHuman epidermal keratinocytesNDLectin receptor-mediated pathway may be involved in cellular uptakeND [66]
SWCNT500–2000 nm1.4 nm (10–40 nm after functionalization)Non-covalent binding with streptavidin-labeled quantum dot and biotinylated anti CD3 antibodyJurkatNDEndocytosis dependent on the interaction of CD3 antigen and its antibodyEndocytic vesicles and not found in a nucleus [67]
SWCNT110 nm10 nmCovalently functionalized (oxidized, EGF, Qdot and/or cisplatin)HN12, HN13, SAA and NIH3T3Selective toxicity dependent on EGFR expressionEGF and EGF receptor-dependent endocytosisPerinuclear region [49]
SWCNT<500 nm1–2 nmCovalently functionalized (oxidized) + acridine orangeHeLaLittle toxicityClathrin-mediated endocytosisLysosomes [68]
SWCNTShort (50–200 nm) or long (200–2000 nm)Small (1–5 nm) or large (3–15 nm)Covalently functionalized (oxidized) + streptavidin or albumin, or non-covalent binding with DNAHeLa and HL60NDClathrin-mediated endocytosisND [52]
MWCNT100–10 000 nm10–15 nmCovalently functionalized (oxidized) + human serum albumin labeled with FITCHepG2 and CRL-4020Selective toxicity by coupling near infrared light and interaction of albumin and gp60-expressing hepatic cancer cellsEndocysosis; co-localization with Gp60 (albumin-binding protein) and caveolin-1Perinuclear localization (as shown in figure) [46]
MWCNT10 000–30 000 nm20–30 nmWith or without covalent functionalization. Nanotubes were dispersed in culture medium containing BSA and DPPCBEAS-2BInduction of IL-1βND (Little uptake of carboxylated nanotubes; nanotubes coated with BSA and DPPC were taken up by the cells whereas non-coated ones were not)Vesicles [43]
MWCNT10 000–40 000 nm50–200 nm (DNA surrounding nanotubes)Non-covalent binding with DNA separately labeled with Cy3 or folateKBNo significant cell death observed up to 4 days incubationND (Possibly via endocytosis because folate-folate receptor interaction facilitates nanotube uptake)ND [69]
SWCNT100–400 nm0.7–32 nmNon-covalent binding with 29-amino acid peptide, folding into an amphiphilic alpha-helix and suspended in culture mediumHeLaLittle toxicityTime- and temperature-dependent fasion (possibly endocytosis)Cytoplasm [70]
SWCNT100–400 nm5–20 nmNon-functionalized and suspended in culture mediumHeLaLittle toxicityTemperature-dependent mechanism (possibly endocytosis)Cytoplasmic vacuoles [71]
SWCNT130–660 nmNDNon-covalent binding with DNANIH3T3NDSize-dependent uptake of nanotubes; hypothesized as receptor-mediated endocytosisND [37]
MWCNT7000–10 000 nm80–130 nmNon-functionalized and suspended in PBS containing 0.1% gelatin, 0.1% CMC or DPPCMESO-1 and BEAS-2BToxicity dependent on dispersant. IL-6 and IL-8 production upon nanotube internalizationNanotubes dispersed in gelatin or DPPC were internalized possibly via endocytosis but nanotubes in CMC were notND [45]
SWCNTSeveral microns0.7–2.1 nmCovalently functionalized (amino group) + plasmid DNAMCF-7Little toxicityHypothesized as endocytosis without experimental evidenceND [72]
Piercing and diffusion
SWCNT300–1000 nm~1 nmCovalently functionalized (amino group + peptide of alpha subunit of Gs protein)3T6 and 3T3Little toxic up to 10 μMPiercing and diffusionCytoplasm and nucleus [54]
SWCNT400 nm~1.4 nmNon-covalent binding with RNAMCF-7Little toxic up to 0.5 mg/mLPiercing and diffusionCytoplasm and nucleus [73]
MWCNT200 nm20 nmCovalently functionalized (amino group) + plasmid DNAHeLa and CHOLittle toxic up to 1.2 mg/mLPiercing and diffusionCytoplasm and nucleus [55]
SWCNTND (commercial)ND (commercial)Covalently functionalized (amino group or FITC)Primary murine immune cells: B cells and T cellsLittle toxicityPiercing and diffusionCytoplasm [53]

SWCNT

and

MWCNT

SWCNT (300–1000 nm) and MWCNT (500–2000 nm)SWCNT (1 nm) and MWCNT (20–30 nm)Covalently functionalized (amino group, acetamido group, FITC, methotrexate and/or amphotericin B)3T6, 3T3, HeLa, Jurkat, keratinocytes, A549, CHO, HEK293 and MOD-KNDPiercing and diffusion, irrespective of functional groups and cell typesPerinuclear region [56]
SWCNTLength-fractionated samples (84–411 nm)ND (commercial)Non-covalent binding with DNAA549, MC3T3-E1, A10 and IMR90Toxicity dependent on length: nanotubes shorter than 189 ± 17 nm are toxicSWCNTs shorter than 189 ± 17 nm enter cells by piercing and diffusionCytoplasm [74]
MWCNTSample 1 (~2000 nm) and sample 2 (~300 nm)Sample 1 (20–40 nm) and sample 2 (>35–40 nm)Non-covalent binding with Pluronic F127 and suspended in cell culture mediumHN9.10e (immortalized mouse hippocampal cells) and PC12NDPiercing and diffusion, judging from the fact that sodium azide and low temperature did not hinder cellular uptakeCytoplasmic vacuoles [75]
MWCNT~1000 nm20–30 nmCovalently functionalized (oxidized or amino group) + FITC-BSAHEK293NDNanotube clusters were taken up by cells via endocytosis and individual ones via piercing and diffusion. Intracellular nanotubes were relatively shortCytoplasm, vesicles and nucleus [76]
MWCNT50–500 nm20–30 nmCovalently functionalized (oxidized followed by introduction of amino groups) and labeled with FITC via π stackingCatharanthus roseus cell (plant cell)Little toxicityPiercing and diffusion (the average lengths of MWCNTs in cells were around 100 nm)Cytoplasm, vacuole, plastids and nucleus [77]

SWCNT

and

MWCNT

SWCNT (~100 nm) and MWCNT (1000–2000 nm)SWCNT (4–5 nm in bundle) and MWCNT (20–30 nm)SWCNT: Covalently functionalized (oxidized) and labeled with FITC via π stackingCatharanthus roseus cell (plant cell)Little toxicityPiercing and diffusion into cytoplasm and active transport into vacuoleCytoplasm and nucleus [78]
MWCNT1000–2000 nm10–30 nmNDA549NDPiercing and diffusion (no negation of endocytic pathway)Cytoplasm and vesicles. Not found in a nucleus [79]

SWCNT

and

MWCNT

SWCNT (50–500 nm) and MWCNT (50–500 nm)SWCNT (0.5–1.5 nm) and MWCNT (10–30 nm)Non-covalent binding with phospholipid polyethylene glycol amineOVCAR8Little toxicityHypothesized as piercing and diffusion without experimental evidenceCytoplasm [80]
MWCNTLargely up to 10 000 nmTangled (12 nm), thin (50 nm) and thick (150 nm)Non-functionalized and suspended in saline containing 0.5% albuminHuman peritoneal mesothelial cells, MeT5A and MDCKIIDiameter- and rigidity-dependent toxicity and carcinogenicity: thin and rigid nanotubes were the most toxic and carcinogenicPiercing and diffusion in a diameter- and rigidity-dependent mannerCytoplasm and nucleus [17]
Entrance mechanism not specified or no evidence of nanotube entry
SWCNTND (commercial), but highly aggregated0.8–1.2 nmNon-functionalized and suspended in culture mediumA549Low acute toxicity (0.015–0.8 mg/mL)No evidence of SWCNT internalization but ultrastructural changes in the cell morphology were observedNo cellular internalization [81]
MWCNT100–13 000 nm12 nmNon-functionalized and suspended in dipalmitoyl lecithin, PBS or ethanolA549 and MeT5ADecrease in metabolic activity without apoptosisSmall indivicdual nanotubes could be internalized but not observed with light or transmission electron microscopyNo cellular internalization [82]
MWCNT (bamboo structure)ND (50 000 nm when synthesized)100 nm (bamboo structure made the thip thinner)Non-functionalized and suspended in culture mediumHuman epidermal keratinocytesSlightly toxic with an induction of IL-8 releaseND (most nanotubes inside cells are <1 μm in length as shown in the figures with a maximal length of 3.6 μm)Cytoplasmic vacuoles [83]
MWCNTNDNDCovalently functionalized (methotrexate and FITC)JurkatNDND (independent from the type of covalently-conjugated functional groups)Perinuclear region [84]
MWCNT10 000–20 000 nm100–150 nmNon-functionalized and suspended in PBS containing 0.1% gelatinBEAS-2BLDH release with IL-6 and IL-8 secretionNDPerinuclear localization [85]
MWCNT300–1000 nm1 nmCovalently functionalized (amino group)A549 and CHOLittle toxicityNDPerinuclear localization [86]
MWCNT500–2000 nm20–30 nmCovalently functionalized (amino group, FITC and/or carboxy group)A549ND (Possibly little toxicity)Both of piercing/diffusion and endocytotic pathway; predominant piercing in the precense of endocytosis inhibitorsPerinuclear localization, cytoplasm and vesicles [87]
MWCNTND (500–2000 nm in ref.)20–30 nmCovalently functionalized (dendron: alkylated amino groups) + siRNAHeLa and A549Little toxic up to 0.08 mg/mLNDPerinuclear localization and cytoplasm [88]
MWCNTShort (100–3500 nm) or long (100–12 000 nm)10–160 nmNon-functionalized and suspended in water containing arabic gumA549Toxic and induces cell deathND (smallest nanotubes with a maximal length of 3 μm were internalized: 500 nm in length and 50 nm in diameter as shown in the figure)Cytoplasm and vesicles; not found in a nucleus [89]
MWCNTND (commercial)ND (commercial)Covalently functionalized (anti-MUC-1 aptamer was conjugated following oxidation)MCF-7 and Calu-6Little toxicityND (nanotubes were taken up by cells irrespective of MUC-1 expression, indicating that the translocation was receptor-independent)Cytoplasm and nucleus (as shown in the figure) [90]
MWCNT900 nmND (<30 nm, judging from the thickness of TEM section)Non-covalent binding with Pluronic F127PC3 and RENCA (a murine renal cancer cell line)Selective toxicity by coupling near infrared lightNDCytoplasmic vesicle and nucleus [91]
MWCNTND (commercial)ND (commercial)Covalently functionalized (oxidized)HepG2NDNDCytoplasm [92]
SWCNT200–400 nm3.8–7.8 nm in ref.Non-covalent binding with PEG; conjugated with CpGGL261Little toxicityNDCytoplasm [93]

SWCNT

and

MWCNT

SWCNT (5000–30 000 nm) and MWCNT (10 000–30 000 nm)SWCNT (1–2 nm) and MWCNT (20–30 nm)Non-functionalized and suspended in PBS containing Tween 80A3, MSTO-211H and HaCaTSWCNTs were more toxic than MWCNTsND (SWCNT penetrating lymphocyte cell membrane is shown in the figure)ND [94]
MWCNT1000–13 500 nm67 nmNon-covalent binding with Pluronic F68BEAS-2B and CHO-K1Toxicity dependent on cell density; various cytokine productionND (No differentiation between engulfed or membrane-bound MWCNT)ND [57]
MWCNT300 nm30–40 nmCovalently functionalized (polyamidoamine) + DNAHeLa and COS-7Low cytotoxicity at low concentration; positive charge of PAA-g-MWCNTs may contribute to the cytotoxicityND (Labeled DNA was localized in perinuclear region)ND [95]

Size and Surface Characteristics of Carbon Nanotubes Determine Uptake Pathways

  1. Top of page
  2. Abstract
  3. Overview of Asbestos-Induced Carcinogenesis
  4. Translocation and Biopersistence of Fibers
  5. Mesothelial and Epithelial Cell Injury Induced by Fibrous Materials
  6. Factors Involved in Carbon Nanotube and Asbestos Fiber Uptake by Non-Phagocytic Cells
  7. Asbestos Fibers are Endocytosed by Mesothelial and Epithelial Cells
  8. Cellular Uptake of Carbon Nanotubes Varies
  9. Size and Surface Characteristics of Carbon Nanotubes Determine Uptake Pathways
  10. Carbon Nanotubes and Asbestos Fibers Activate Macrophages
  11. Concluding Remarks
  12. Acknowledgments
  13. Disclosure Statement
  14. References

Size-dependent uptake of carbon nanotubes into cells has been well studied.[17, 37, 38] Kang et al.[38] showed that SWCNTs, but not MWCNTs, all of which were oxidized and non-covalently bound with chitosan, mainly entered human HepG2 hepatocellular carcinoma cells via endocytosis. This result indicates that large carbon nanotubes cannot be taken up by cells through endocytosis due to size limitations. However, it is notable that the conjugation of folic acid on MWCNTs enabled them to enter cells, indicating that specific, receptor-mediated endocytosis allows large fibers to be internalized.[38-42] We recently showed that MWCNTs coated with albumin on their surface were not endocytosed by two different human mesothelial cell lines.[17] Instead, we found that these MWCNTs directly pierced through the plasma and nuclear membranes in a diameter- and rigidity-dependent manner, suggesting that large MWCNTs can still enter cells through direct piercing rather than endocytosis. On the other hand, Wang et al.[43] showed that MWCNTs coated with both albumin and phospholipid (DPPC, dipalmitoyl-phosphatidylcholine) were successfully internalized by bronchial epithelial BEAS-2B cells. Moreover, Haniu et al.[44, 45] showed that MWCNTs bound to gelatin or a phospholipid (DPPC) were taken up by mesothelioma MESO-1 cells, bronchial epithelial BEAS-2B cells and neuroblastoma IMR-32 cells. Taken together, current data indicate that MWCNTs, which are comparatively larger than endosomes, may not be endocytosed, but the presence of specific molecules, such as folic acid and DPPC, may help cells take up MWCNTs. Albumin did not facilitate endocytosis in our previous study, but previous reports indicate that albumin may help endocytosis if the target cells express high levels of an albumin receptor, such as gp60.[46] Similarly, Jin et al.[37] showed that among small SWCNTs, the efficiency of nanotube uptake is also size-dependent. They showed that SWCNTs wrapped by DNA were most efficiently taken up by NIH3T3 cells when the nanotube length was ~300 nm.

Regarding the size limitation on membrane piercing, it is of note that Kusumi and colleagues reported a membrane skeleton fence model.[47, 48] This model explains that an actin mesh network exists just beneath the cell membrane to form compartments whose size are dependent on cell type (side length is 30–230 nm). Based on this model, we can expect that very thick nanotubes (>230 nm) that evade endocytosis due to their surface property cannot directly pierce the membrane.

Surface characteristics are also critical in carbon nanotube uptake. For instance, oxidized nanotubes are more likely to enter cells via endocytosis with the help of specific ligands[42, 46, 49-52] because the oxidation of nanotubes introduces carboxyl groups, which provides a negative charge to their surface, resulting in repulsion from the plasma membrane unless the carbon nanotube has ligands that are recognized with a high affinity by the cell. Many ligands (folic acid,[42] albumin[46] and epidermal growth factor[49]) have been reported to facilitate uptake by specific cell types. Alternatively, a carbon nanotube with amino groups presents a positive charge on its surface and can enter cells via energy-independent piercing and diffusion.[53-55] On the other hand, Kostarelos et al.[56] showed that MWCNTs with functional groups entered various types of cells in a functional group-independent manner via membrane piercing and diffusion. In our own studies we have found that pristine MWCNTs with albumin on their surface pierced the cell membrane in a diameter-dependent manner.[17] These results indicate that a positive charge on the surface of carbon nanotubes might not be necessary for the carbon nanotubes to pierce through the cell membrane. However, the detailed mechanism remains to be elucidated.

Carbon Nanotubes and Asbestos Fibers Activate Macrophages

  1. Top of page
  2. Abstract
  3. Overview of Asbestos-Induced Carcinogenesis
  4. Translocation and Biopersistence of Fibers
  5. Mesothelial and Epithelial Cell Injury Induced by Fibrous Materials
  6. Factors Involved in Carbon Nanotube and Asbestos Fiber Uptake by Non-Phagocytic Cells
  7. Asbestos Fibers are Endocytosed by Mesothelial and Epithelial Cells
  8. Cellular Uptake of Carbon Nanotubes Varies
  9. Size and Surface Characteristics of Carbon Nanotubes Determine Uptake Pathways
  10. Carbon Nanotubes and Asbestos Fibers Activate Macrophages
  11. Concluding Remarks
  12. Acknowledgments
  13. Disclosure Statement
  14. References

Macrophages are a type of immune cell that function in the recognition and removal of pathogens and exogenous materials. Macrophages sometimes fail to achieve this goal when the pathogens are too large and/or resistant to biodegradation. Certain nanotubes and asbestos fibers have these features, leading to persistent macrophage activation and enhanced chronic inflammation.[5, 6]

It has been reported that macrophages recognize asbestos fibers and carbon nanotubes via the class A scavenger receptor, MARCO (macrophage receptor with collagenous structure).[57-60] Moreover, the uptake of these two types of materials activates the NLRP3 inflammasome.[61, 62] Therefore, asbestos fibers and carbon nanotubes appear to activate macrophages through similar mechanisms. It is noteworthy that the carbon nanotubes used in these experiments were non-functionalized.

Concluding Remarks

  1. Top of page
  2. Abstract
  3. Overview of Asbestos-Induced Carcinogenesis
  4. Translocation and Biopersistence of Fibers
  5. Mesothelial and Epithelial Cell Injury Induced by Fibrous Materials
  6. Factors Involved in Carbon Nanotube and Asbestos Fiber Uptake by Non-Phagocytic Cells
  7. Asbestos Fibers are Endocytosed by Mesothelial and Epithelial Cells
  8. Cellular Uptake of Carbon Nanotubes Varies
  9. Size and Surface Characteristics of Carbon Nanotubes Determine Uptake Pathways
  10. Carbon Nanotubes and Asbestos Fibers Activate Macrophages
  11. Concluding Remarks
  12. Acknowledgments
  13. Disclosure Statement
  14. References

A number of sequential events are involved in fibrous material (i.e. carbon nanotubes and asbestos fibers) toxicology: translocation of materials, biopersistence of materials, mesothelial/epithelial cell injury induced by materials and macrophage activation triggered by materials. Among these, we focused on how asbestos fibers and carbon nanotubes enter non-phagocytic cells, which is important in mesothelial/epithelial cell injury.[96, 97] Asbestos fibers are endocytosed by these non-phagocytic cells and carbon nanotubes behave variably according to their functionalization and size (Fig. 2). Based on current evidence, a positive charge on the surface and a thin diameter appear to be two important factors in facilitating the membrane piercing ability of carbon nanotubes. The presence of specific ligands on the carbon nanotube surface would induce ligand-mediated endocytosis, which allows large-sized nanotubes to enter non-phagocytic cells. Further investigation regarding the mechanism of entry of carbon nanotubes and asbestos fibers into non-phagocytic cells is warranted to establish the distinct toxicological paradigms of carbon nanotubes and asbestos fibers.

image

Figure 2. A schematic of the fiber entry mechanism into a non-phagocytic cell. Asbestos fibers are endocytosed by the cells through phagocytosis-like mechanisms. Mechanisms of carbon nanotube uptake depend on the characteristics of each fiber. Nanotubes with thin diameter (~50 nm in Fig. 1), high rigidity and positive surface charge are likely to pierce the plasma and nuclear membrane whereas nanotubes with shorter length (<1 μm) and specific ligands on their surface with modification would be endocytosed by the cells.

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Acknowledgments

  1. Top of page
  2. Abstract
  3. Overview of Asbestos-Induced Carcinogenesis
  4. Translocation and Biopersistence of Fibers
  5. Mesothelial and Epithelial Cell Injury Induced by Fibrous Materials
  6. Factors Involved in Carbon Nanotube and Asbestos Fiber Uptake by Non-Phagocytic Cells
  7. Asbestos Fibers are Endocytosed by Mesothelial and Epithelial Cells
  8. Cellular Uptake of Carbon Nanotubes Varies
  9. Size and Surface Characteristics of Carbon Nanotubes Determine Uptake Pathways
  10. Carbon Nanotubes and Asbestos Fibers Activate Macrophages
  11. Concluding Remarks
  12. Acknowledgments
  13. Disclosure Statement
  14. References

We thank Shan Hwu Chew (Nagoya University) for her critical comments and suggestions on the manuscript. This study was supported by Princess Takamatsu Cancer Research Fund Grant 10-24213; grant-in-aid for Cancer Research from the Ministry of Health, Labor and Welfare of Japan; a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan; a MEXT Special Coordination Funds for Promoting Science and Technology Grant; a grant from the Takeda Science Foundation; and a grant-in-aid from the Japan Society for the Promotion of Science Fellows (HN).

References

  1. Top of page
  2. Abstract
  3. Overview of Asbestos-Induced Carcinogenesis
  4. Translocation and Biopersistence of Fibers
  5. Mesothelial and Epithelial Cell Injury Induced by Fibrous Materials
  6. Factors Involved in Carbon Nanotube and Asbestos Fiber Uptake by Non-Phagocytic Cells
  7. Asbestos Fibers are Endocytosed by Mesothelial and Epithelial Cells
  8. Cellular Uptake of Carbon Nanotubes Varies
  9. Size and Surface Characteristics of Carbon Nanotubes Determine Uptake Pathways
  10. Carbon Nanotubes and Asbestos Fibers Activate Macrophages
  11. Concluding Remarks
  12. Acknowledgments
  13. Disclosure Statement
  14. References