Grantee Research Project Results
Final Report: Physical and Chemical Determinants of Nanofiber/Nanotube Toxicity
EPA Grant Number: R831719Title: Physical and Chemical Determinants of Nanofiber/Nanotube Toxicity
Investigators: Hurt, Robert H. , Kane, Agnes B.
Institution: Brown University
EPA Project Officer: Hahn, Intaek
Project Period: August 1, 2004 through July 31, 2007
Project Amount: $335,000
RFA: Exploratory Research to Anticipate Future Environmental Issues: Impacts of Manufactured Nanomaterials on Human Health and the Environment (2003) RFA Text | Recipients Lists
Research Category: Nanotechnology , Human Health , Safer Chemicals
Objective:
Carbon nanotubes (CNTs) and related fibrous materials play a very special role in emerging nanotechnologies. The early nanotoxicology literature contains conflicting reports on the biological response to carbon nanotubes, and we hypothesize that some of the variability is due to real sample-to-sample differences in material properties. Most commercial nanotubes or nanofibers are complex materials containing amorphous carbon, transition metal catalysts or residues and tubular material with complex distributions of length and diameter, as well as variability in graphene defect density and surface functional groups. The objective of this project is to carry out a carefully designed parametric study of the physical and chemical factors that underlie nanofiber/tube toxicity, in which the effects of shape, size, surface chemistry, and especially metals content are carefully isolated by special synthesis and characterization techniques developed at Brown University.
Summary/Accomplishments (Outputs/Outcomes):
This project provided important data on the fundamental factors responsible for the adverse biological responses to carbon nanotubes. The early work in nanotoxicology did not recognize the complex nature of carbon nanotube samples, and in particular the potential for metal-induced toxicity. By systematic study of metal release from nanotubes under physiologically relevant conditions, the presence of toxicologically significant quantities of bioavailable nickel and iron was documented in both as-produced and vendor “purified” nanotubes from a variety of commercial sources. The metal release was found to be sensitive to environmental exposure and to nanotube material stresses during common processing operations. These results suggest methods for mitigating the adverse health impacts of nanotubes through more intelligent fabrication, purification, and processing. It was also shown, for the first time, that the large hydrophobic surface area of nanotubes causes a depletion in essential micronutrients in cell culture medium, most notably folic acid, and this depletion can lead to an inhibition of cell proliferation and apparent toxicity to cells in culture. This is a fundamentally new and indirect mechanism that does not require physical contact between the nanotubes and any living matter. Finally, in the last stages of the project, we explored the use of an antioxidant surfactant derived from Vitamin E as a novel method for green processing and potential toxicity mitigation in carbon nanotubes and fullerenes. These four major findings (Fe bioavailability and redox activity, Ni bioavailability and cell uptake, folate depletion, and the use of a new antioxidant surfactant for green processing) led to four archival publication in high-impact journals as described in more detail in separate sections below. The work has also been presented through a variety of invited and contributed talks, some given by graduate students at major international meetings (see below).
Bioavailability and redox activity of nanotube iron
Carbon nanotubes are now primarily fabricated by catalytic routes and typically contain significant quantities of transition metal catalyst residues, including iron. Iron-catalyzed free radical generation has been hypothesized to contribute to oxidative stress and toxicity upon exposure to ambient particulate, amphibole asbestos fibers, and most recently single-wall carbon nanotubes. A key issue surrounding nanotube iron is bioavailability, which has not been systematically characterized, but is widely thought to be low based on electron microscope observations of metal encapsulation by carbon shells. In this project, simple acellular assays were validated and applied to show that toxicologically significant amounts of iron can be mobilized from a diverse set of commercial nanotube samples in the presence of ascorbate and the chelating agent ferrozine. This mobilized iron is redox active and induces single strand breaks in plasmid DNA in the presence of ascorbate. Iron bioavailability varies greatly from sample to sample and cannot be predicted from total iron content. Iron bioavailability is not fully suppressed by vendor “purification” and is sensitive to partial oxidation, mechanical stress, sample age, and intentional chelation. For these reasons it is not sufficient in nanotoxicology studies to identify CNT samples only by the labels “as-produced” or “purified,” nor to report total iron by standard elemental analysis. A specific assay for free, or bioavailable, iron is necessary for the complete characterization of CNTs used in biological applications or nanotoxicology studies. The extent of iron release represents from 1% to 7% of the total iron, which is up to half of the iron mobilized from crocidolite asbestos used as a reference material. These results help reconcile two opposing viewpoints on CNT metals. The first viewpoint, held by some researchers involved in CNT synthesis and manufacturing, is that the metals are fully encapsulated - a viewpoint that is based on electron microscopy as well as the common observation that oxidation is required to damage the carbon shells before significant quantities of metal can be removed by acid treatment. The second viewpoint, held by some toxicologists, is that iron must be bioavailable to explain Fe-induced oxidative stress and lipid peroxidation observed in recent cell culture studies. The present results make it clear that most of the iron is indeed encapsulated (93% - 99% not mobilized in a 3-day assay in the presence of strong chelators), but a small fraction is fluid accessible and that fraction is sufficient to release toxicologically relevant amounts of iron and drive redox reactions that cause DNA single strand breaks. The present results also suggest that CNT sample age is an important variable in toxicity. A custom synthesized Fe/C-nanofiber model system shows dramatic variation in Fe-mobilization over the course of one-year atmospheric exposure due to progressive oxidation. We anticipate that the small fraction of iron that is accessible in catalytically grown nanotubes undergoes a similar aging process and that this heretofore undocumented aging effect can lead to complex and time-dependent CNT toxicity profiles that may contribute to variability and inconsistency between data sets in the current CNT toxicology literature.
Bioavailability of nickel in single-wall carbon nanotubes
Another important CNT growth catalyst is nickel, which is the majority component in Ni-Y catalysts commonly used in the arc synthesis of single-wall nanotubes (SWNTs). Nickel is also an established human carcinogen that induces gene silencing and hypoxia signaling through mechanisms involving intracellular nickel cation. It is not known whether carbon nanotube catalyst residues can trigger these toxicity mechanisms due to apparent encapsulation of the nickel within carbon shells. This project produced the first systematic measurements of Ni bioavailability. The data show that, despite apparent encapsulation, toxicologically significant quantities of nickel are released from a range of single-wall nanotube samples and can be transported across the plasma membrane into human lung epithelial cells. The nickel release varies greatly as a function of nanotube source and processing history. Nickel bioavailability varies greatly from sample to sample in a manner that cannot be predicted from total elemental analysis, and thus here also a new, dedicated assay is required. A variety of assays were evaluated for nickel bioavailability, and a simple acellular assay was developed and tested that is suitable as a standard biologically-relevant nanomaterials characterization method.
In addition, detailed TEM examination provided some insight into the mechanism of Ni release. Acid washing causes the appearance of a few completely hollow carbon shells, but leaves most of the Ni nanoparticles fully intact, even those covered only by ultra-thin carbon shells. This fact strongly suggests that carbon shells are not intrinsically permeable to nickel, but rather the process of Ni release is driven by defects in the shells that are not readily seen in two-dimensional projection. Finally, we show that Ni bioavailability is sensitive to nanotube processing, and thus there is great potential to reduce SWNT health risks by more intelligent management of the imbedded nickel residues, including targeted removal of the bioavailable fraction in a manner that does not introduce additional defects in the carbon shells and expose fresh metal.
Adsorption of essential micronutrients by nanotubes in cell culture
Nanotoxicology is a new field that now relies on in vitro cellular assays that were developed prior to the nanotechnology era. The introduction of nanomaterials to these standard assays causes problems that are currently limiting progress in the field. Nanoparticles are often difficult to disperse, they can interfere with optical measurements through light adsorption, and they can interact with dyes used as molecular probes of cellular integrity. In some cases the resulting artifacts can lead to gross misinterpretation of effects on cell viability and cytotoxicity. Because sp2-hybridized carbon materials are near-universal sorbents for organic compounds in aqueous phases, it was hypothesized here that single-wall carbon nanotubes would adsorb a wide variety of small organic solutes from biological media, not limited to indicator dyes or their water-insoluble reduction products. Biochemical profiling techniques and UV/visible spectroscopy were used to show that SWNTs cause dose-dependent adsorption and depletion of over 14 amino acids and vitamins from RMPI cell culture medium. HepG2 cells cultured in these depleted media show significantly reduced viability.
We have found that folic acid is strongly depleted at nanotube doses as low as 0.01 mg/ml. Folate deficiency is known to have deleterious effects on DNA metabolism and this deficiency is exacerbated by deficiency of other vitamins including riboflavin. In proliferating cells in culture, short-term folate deficiency is known to reduce cell proliferation, induce cell cycle arrest, trigger apoptosis, and alter expression of microRNAs associated with stress responses in primary human lymphocytes as well as lymphoblastoid and hepatocyte cell lines. To test the specific role of folate depletion, a variety of the nanotube-depleted media were supplemented with external folate and a reversal of the effect on cell viability was observed.
In summary, single-wall carbon nanotubes have sufficient surface area to alter the micronutrient content of cell culture medium through adsorption of small molecule solutes at CNT doses as low as 0.01 mg/ml. Because physical adsorption is only weakly specific, this is a general phenomenon that influences a broad spectrum of small molecule solutes including amino acids, vitamins, and indicator/probe dyes. The extent of adsorption is greatest for solutes at low initial concentrations and having molecular structure favoring adsorption on graphenic carbon – hydrophobicity, planarity/sp2-hydridization for π-π interactions, and positive charge. The depletion of folate, as well as other essential micronutrients at doses of 0.01–0.1 mg/ml significantly reduced HepG2 cell viability by an indirect mechanism that does not involve physical nanotube/cell interaction. An important implication of this finding is that CNT toxicity, as assessed by commonly-used endpoints such as cell viability, DNA damage, and apoptosis, may be mistakenly attributed to direct toxicity, when in fact it is a secondary effect of adsorption of essential micronutrients from cell culture medium.
TPGS as a safe, antioxidant surfactant for nanotube processing
The project also investigated the physical interactions between carbon nanomaterials and tocopheryl polyethylene glycol succinate (TPGS). TPGS is a synthetic amphiphile that undergoes enzymatic cleavage to deliver the lipophilic antioxidant, a-tocopherol (vitamin E) to cell membranes, and is FDA approved as a water-soluble vitamin E nutritional supplement and drug delivery vehicle. It was shown that TPGS 1000 is capable of dispersing multi-wall and single-wall carbon nanotubes in aqueous media, and for multiwall tubes is more effective than the commonly used non-ionic surfactant Triton X-100. TPGS is also capable of solubilizing C60 in aqueous phases by dissolving fullerene in the core of its spherical micelles. Drying of these solutions leads to fullerene/TPGS phase separation and the self-assembly of highly ordered asymmetric nanoparticles, with fullerene nanocrystals attached to the hydrophobic end of crystalline TPGS nanobrushes.
Overall, TPGS is a promising surfactant for “green” processing of carbon nanomaterials due to its low human health risk, commercial availability, and effectiveness as a dispersant and stabilizer. TPGS is especially promising for multi-wall nanotube processing, where it is an effective dispersant at mass ratios (TPGS:C) of 1:4 or greater and is more effective than the commonly used synthetic non-ionic surfactant Triton. In light of the present results on the physical interactions of TPGS with carbon nanomaterials, detailed biological studies are justified to determine if and under what conditions the antioxidant properties of TPGS may serve to actively mitigate oxidative stress induced by nanomaterial exposure by delivering Vitamin E to the cell membrane locally at the site of nanomaterial/cell contact.
Other work
Finally during the last project year we began to explore methods for detoxifying nanotubes through targeted removal of the bioavailable portion of the total metal, making use of the new understanding of metal location, form, and release behavior in physiological fluids. This work is continuing beyond the current project, and should lead to practical procedures for nanotube purification that target the bioavailable metal and are more effective than current purification processes in reducing nanotube health risks.
Overall, this project produced new insights into the underlying causes of nanotube toxicity, and has explored new methods of purifying, handling, or formulating nanotubes to reduce nanotube health risks.
Journal Articles on this Report : 7 Displayed | Download in RIS Format
| Other project views: | All 24 publications | 7 publications in selected types | All 7 journal articles |
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Guo L, Morris DG, Liu X, Vaslet C, Hurt RH, Kane AB. Iron bioavailability and redox activity in diverse carbon nanotube samples. Chemistry of Materials 2007;19(14):3472-3478. |
R831719 (Final) |
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Guo L, Liu XY, Sanchez V, Vaslet C, Kane AB, Hurt RH. A window of opportunity: designing carbon nanomaterials for environmental safety and health. Materials Science Forum 2007;544-545:511-516. |
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Guo L, Von Dem Bussche A, Buechner M, Yan A, Kane AB, Hurt RH. Adsorption of essential micronutrients by carbon nanotubes and the implications for nanotoxicity testing. Small 2008;4(6):721-727. |
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Hurt RH, Monthioux M, Kane A. Toxicology of carbon nanomaterials: status, trends, and perspectives on the special issue. Carbon 2006;44(6):1028-1033. |
R831719 (2006) R831719 (Final) |
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Liu X, Gurel V, Morris D, Murray DW, Zhitkovich A, Kane AB, Hurt RH. Bioavailability of nickel in single-wall carbon nanotubes. Advanced Materials 2007;19(19):2790-2796. |
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Sanchez V, Pietruska J, Meselis N, Hurt R, Kane A. Biopersistence and potential adverse health impacts of fibrous nanomaterials: what have we learned from asbestos. NANOMEDICINE AND NANOBIOTECHNOLOGY 2009;1(5):511-529 |
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Yan A, Von Dem Bussche A, Kane AB, Hurt RH. Tocopheryl polyethylene glycol succinate as a safe, antioxidant surfactant for processing carbon nanotubes and fullerenes. Carbon 2007;45(13):2463-2470. |
R831719 (Final) |
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Supplemental Keywords:
health effects, human health, carcinogen, cellular, toxics, particulates, metals, oxidants, environmentally conscious manufacturing, nanotechnology, engineering, pathology, industry, health, sustainable industry/business, biochemistry, environmental chemistry, environmental engineering, health risk assessment, risk assessments, sustainable environment, technology for sustainable environment, asbestos like toxicity, biochemical research, carbon fullerene, engineered nanomaterials, exposure, genotoxins, human exposure, human health risk, nanofiber, nanofibers, nanotechnology, nanotube toxicity,, Sustainable Industry/Business, RFA, Health, Scientific Discipline, Technology for Sustainable Environment, Health Risk Assessment, Risk Assessments, Sustainable Environment, Environmental Chemistry, Biochemistry, human exposure, nanotube toxicity, engineered nanomaterials, nanotechnology, nanofiber, exposure, nanofibers, asbestos like toxicity, human health risk, genotoxins, particle exposure, biochemical researchRelevant Websites:
http://www.engin.brown.edu/Facilities/LINC/ Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.