Toxicity of Carbon Nanotubes

1. Toxicity of Carbon Nanotubes Aziz Daabash Brett Michalk Amanda Mogollon Derek Nelson

2. Outline • Introduction • Types • Properties • Toxicity • Respiratory • Reactivity • Effect on humans • Conclusions • Recommendations • Further Research http://www.aip.org/png/images/ando.jpg

3. Introduction A carbon nanotube (CNT) is a tubular molecule with axial simmetry and diameter in the nanometer range (Muller). It can be considered as a rolled up graphene sheet. However, it possesses many properties that leave no doubt this is not just graphene. http://www.msm.cam.ac.uk/phasetrans/2005/paper/img19.png http://www.nanotech-now.com/nanotube-buckyball-sites.htm

4. Types • Single Walled CNT (SWCNT): one-atom-thick CNTs • Multi Walled CNT (MWCNT): concentric layers of CNTs http://www-ibmc.u-strasbg.fr/ict/images/SWNT_MWNT.jpg

5. Properties Among some of the properties of the CNTs we can find: • Electrical: Both metallic and non-metallic behaviors are observed, while geometry plays a profound part in determining the electronic behavior. (Ebbesen) • Elastic: Tensile Young’s module and torsion shear module comparable to that of diamond (Lu). http://www.studentsoftheworld.info/sites/family/img/27335_Electricity.jpg http://www.nanoshel.com/research-center/wp-content/uploads/2009/01/ballistic-impact.jpg

6. Properties • Mechanical: Carbon nanotubes have high strength plus extraordinary flexibility and resilience. (Salvetat) • Thermal: Thermal expansion of carbon nanotubes will be essentially isotropic that is, uniform in all directions (Ruoff). http://brent.kearneys.ca/wp-content/uploads/2006/05/carbon_nanotube.jpg

7. Toxicity Due to the remarkable range of properties that carbon nanotubes posses, they have numerous applications and can be used in a variety of industries that range from electronics to food processing. Since CNTs may be present in our every day lives in a matter of a few years, a concern on its toxicity has been growing recently. http://www.artsandopinion.com/2007_v6_n6/volume_images/toxic-1.jpg

8. Toxicity Why is it important to determine CNTs toxicity? • CNTs manufacturers and suppliers, like Carbon Nanotechnologies, Inc in Houston, Texas, classified CNTs as synthetic graphene and using it as a reference for the permissible inhalation and exposure limit (PEL) (Lam). http://www.lifemedicalsupplier.com/images/mask_1860.jpg

9. Toxicity • However, it is clear that CNTs do not have the same properties as graphene, and thus this PEL might not reflect the real toxicity and exposure limits of CNTs. http://www.tcd.ie/Chemistry/staff/people/duesberg/ASIN%20web%2027-10-09/images/CNT.jpg http://cnx.org/content/m29187/latest/graphene.jpg

10. Julie Muller, François Huaux, Dominique Lison Respiratory toxicity of carbon nanotubes: How worried should we be? *Note: All materials in the following section are obtained from the above research paper.

11. Introduction Some physical characteristics of nanotubes, like their length to diameter ratio, low solubility in aqueous media, propensity to agglomerate, being light and able to become airborn, among others, suggest that they may be toxic. http://romunov.blogsome.com/images/kozarec.jpg

12. Introduction Residual catalytic material like iron, cobalt, and nickel, left from different manufacturing or purification processes may contribute to the toxicity of CNTs. http://www.mcgill.ca/files/_nea/106127_labSmall.jpg

13. Introduction Inhalation exposures in industrial settings should be very low, but since there is little information on the respiratory toxicity of CNTs, it cannot be concluded that the risk is negligible. http://images.lifescript.com/images/ebsco/images/inhaled_poison.jpg

14. Are CNTs toxic? • In order to answer this question, the authors gathered information from other experiments and compared results • Noticed a number of parameters that could have affected the results of some experiments • Non-grinded CNTs tend to agglomerate • Concentration of metal impurities http://public.blu.livefilestore.com/y1pxaiFNCiK21KETuwhBtq0QlnoQQq9MkqWxmJhOOPK1PJ6ZMg7VstXtxs3Er1fERLLuQeBN4mCM5XiOx7XK15HHw/metales.jpg

15. Experiment Comparison Muller et al.

16. Results • Non-grounded MWCNTs agglomerate in large airways, while ground ones disperse in the lungs. • When MWCNTs reach the lungs, they are not rapidly cleared. Intact MWCNTs are retained longer than ground ones. http://4.bp.blogspot.com/_vfImvyorvjQ/Sxr8EMfTESI/AAAAAAAAAbQ/gHWF8X3ToxI/s320/arkema+1.jpg

17. Results Sections of control rat lung (A), and immediately after intratracheal instillation of 2 mg/animal ground MWCNTs (C, arrows) which are much better dispersed in the lung than intact MWCNTs which mainly remained clumped in large airways (B, arrows). Muller et al.

18. Results • The lung toxicity of CNTs was of similar or greater intensity than well-recognized lung toxicants such as quartz or crocidolite fibers. • Transition metals left as impurities might contribute to the toxicity of CNTs. Crocidolite Quartz http://www.isitasbestos.com/images/crocidolite.jpg http://www.windows2universe.org/earth/geology/images/3quartz.JPG

19. Conclusions • If they reach the lungs, CNTs (SWCNTs and MWCNTs) have potential to cause severe inflammatory and fibrotic reactions.

20. Further Research • CNTs should be tested in other animals to determine the different toxic behaviors. • Animals should be placed in an environment in which CNTs are present, instead of placing the CNTs intratracheally, to determine if they can reach the lungs when inhaled. http://www.dichotomistic.com/images/particles%20in%20box.jpg

21. Reactivity of carbon nanotubes: Free radical generation or scavenging activity? IvanaFenoglio a, Maura Tomatis a, Dominique Lison b, Julie Muller b, Antonio Fonseca c, Janos B. Nagy c, BiceFubini Source: http://nanotechnologytoday.blogspot.com/2009_11_01_archive.html *Note: All materials in the following section are obtained from the above research paper.

22. Introduction When passing from micro to nano scale, most materials undergo remarkable modifications in their chemical properties, often associated with an increase in their reactivity. Because of their unique properties, These properties make CNT suitable for many industrial applications including biomedical applications. Dividing osteoblast on multi-walled CNTs(Source: Dr. Laura Zanello, University of California-Riverside)

23. Potential toxicity When inhaled, CNTs may constitute a possible hazard to the health of exposed workers or users. The determinants of the toxicity of inhaled particles involve three main factors: • form of the particle (e.g., fibrous vs. isometric and smooth). • surface reactivity, typically the potential to generate free radicals. • persistence in the respiratory tract (low solubility and/or slow clearance). Morphology changes of MSTO-211H cells after 3 days of exposure to 15 µg/ml of different fractions of CNTs and asbestos. (a) Untreated MSTO-211H cell culture and (b) cell culture exposed to asbestos. Arrows indicate needles of asbestos. (c) Cells treated with CNT agglomerates were round-shaped and lost their adherence on the cell culture plate. Arrow point to CNT agglomeration. (d) Cells exposed to CNT-bundles showed no visible morphological changes compared to the control cells. (e) Effect of CNT-pellet fraction. Non-tubes material agglomerated during the incubation period to micro-sized structures. (f) Cells incubated with CNT raw material. Source: www.nanowerk.com/spotlight

24. Reactivity Recent researches reported that purified MWCNT are active in inducing inflammatory and fibrotic reactions in the lung in a rat model. Such reactions could contribute to the generation of reactive oxygen species (ROS). ROS may react with extracellular fluids and cellular products or directly damage target cells. Sources :Muller et al.

25. Reactivity (Cont.) This report investigates whether the adverse biological response to nanotubes reported in some studies could be caused by surface reactivity. How? By examining the potential of these materials to generate in aqueous suspension HOS or carbon-centered radicals from hydrogen peroxide and formate ions. Such reactions did not take place, the report subsequently investigated whether nanotubes may act as scavengers of oxygen-centered free radicals. Goal

26. Investigation Unpurified SWCNT, containing a large amount (30%) of iron, were able to stimulate and induce the production of ROS from human keratinocyte cells. It has already been reported that nanotubes exhibit antioxidant properties in nanotube polymer composites. Ex. Fullerenes (form of crystalline carbon) Fullerenes were recently reported to be cytotoxic to human skin and liver cells, the toxic effect being related to the generation of ROS, which damage cell membranes Fullerenes Source: http://www.surf.nuqe.nagoya-u.ac.jp/nanotubes/omake/fullerenes/fullerenes-e.html

27. Goal of Investigation The scavenging activity of nanotubes was tested for the hydroxyl radical and superoxide anion (O2*). hydroxyl radical is the most reactive radical among ROS and the major inducer of oxidative stress in pathological processes What is oxidative stress ? stress caused by an imbalance between the production of reactive oxygen and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage What is superoxide anion (O2 *)? A relatively stable radical which is involved in both pathological and physiological processes.

28. Materials • Multiwall carbon nanotubes • Multiwall carbon nanotubes (MCNT) were synthesized by the decomposition of ethylene on an alumina support doped with a cobalt–iron catalyst mixture and purified by subsequent treatment with NaOH. • MCNT were grounded in an oscillatory agate ball mill (Pulverisette 0, Fritsch) with a vertical vibration of 1 mm applied for 6 hours. • Properties of purified MCNT: • Small amounts of Co (0.29%), Fe (0.47%), and Al (0.05%). • OD= 9.7 ± 2.1 nm mean length =5.9 ± 0.05 μm • BET surface area = 378 m2/g , carbon content = 97.8%. Multiwall carbon nanotubes http://www.msm.cam.ac.uk/Department/DeptInfo/StaffProfiles/ResearchFigs/Windle.jpg 5,5-Dimethyl-1-pyrroline N-oxide Along with the following materials: www.chemblink.com/structures/3317-61-1.gif DMPO (5,5-dimethyl-1-pyrroline-Noxide) Pyrogenic amorphous silica Aerosil 300 Min-U-Sil 5 quartz

29. Methods 1)- Free radical generation (HO*) radicals: Generated by suspending 5 mg of MWCNT in 50 μl of 5% sodium dodecyl sulfate (SDS) diluted in 500 μl of 0.2 M potassium phosphate buffer, pH 7.4, containing 75 mM DMPO and 80 mM hydrogen peroxide. (CO2*) radicals: Generated by suspending of 5 mg of MWCNT in 50 μl of 5% SDS diluted in 625 μl of a buffered 0.2 M potassium phosphate buffer, pH 7.4, containing 75 mM DMPO and 1 M sodium formate. 2)- Scavenging activity toward hydroxyl radicals Hydroxyl radicals (HO*) generated by adding 250 μl of a 0.2 M solution of hydrogen peroxide in water to a solution containing 0.025 M DMPO and 1.7 mM FeSO4 in 0.17 M potassium phosphate buffer, pH 7.4. The yield of HOS radicals was followed by measuring the intensity of the spectrum of the DMPO/HOS adduct. 3)- Scavenging activity toward superoxide radicals Superoxide radicals (O2*-) were generated in a buffered solution (1.2 mM phosphate buffer, pH 7.8) containing 2.4 Μm EDTA, the enzyme xanthine oxidase (0.017 U/ml), and at two different concentrations of xanthine. free radicals released from CNT in was monitored by ESR spectroscopy with DMPO as trapping agent.

30. Results 1)- Free radical generation by multiwall carbon nanotubes Safe Under none of the conditions tested, however, did nanotubes produce any detectable radicals. Figure 1

31. Results (Cont.) 2)- Scavenging activity of multiwall carbon nanotubes • Hydroxyl radicals were generated by the Fenton reaction: • H2O2 þ Fe2þ → HOS þ OH þ Fe3þ • Tested reaction conditions for radicals generated by the Fenton reaction : • Case (a): generation of HOS radicals by the Fenton reaction only. • Case(c): presence of a suspension of 5mg of nanotubes in 50 μl of 5% SDS, signal was suppressed. • Case(b): presence of a high surface area amorphous silica, signal wasn’t effected. Indicates that the scavenging effect is not due to the presence of small particles with a large surface area, but to a more specific reaction ascribed to MWCNT

32. Results (Cont.) To investigate if the observed effect was due to a scavenging property toward radicals or to the inactivation of iron ions (HO*) radicals were also generated by photolysis of hydrogen peroxide: H2O2 → 2HOS Tested reaction conditions for radicals generated by the above photolysis reaction, note the linear increase of radicals produced increased with the concentration of (Figure1). Figure.1 Figure.1

33. Results (Cont.) • Tested reaction conditions for radicals generated by the above photolysis reaction (Figure2): • Case (a): generation of HOS radicals by the photolysis of hydrogen peroxide. • Case(c): presence of a suspension of nanotubes in 5% SDS, signal was completely suppressed. • Case(b): presence of SDS alone, signal was slightly effected. Verifies that the surfactant SDS did not substantially interfere with the radical yield Figure.2

34. Results (Cont.) Scavenging activity of multiwall carbon nanotubes on the superoxide radical • Superoxide radicals (O2*) : • Generated by the xanthine . • Detected by spectrophotometry (monitoring the reduction of cytochrome). • Effect of MWCNT on superoxide radicals at two different concentrations of xanthinein(Figure.1): • Absence MWCNT (light gray bars) • Presence of MWCNT (dark gray bars) Verifies that MWCNT doesn’t have any effect on the concentration of superoxide anions Figure.1

35. Results (Cont.) Superoxide radicals (O2*) were also: Generated by irradiating with a UV lamp a solution of riboflavine and DMPO in phosphate buffer (pH 7.4) located in the spectrometer cavity. • Case (a): The generation of (O2*) by the decomposition of its adduct. • Case(b): presence of MWCNT in 5% SDS, signal almost completely suppressed. • Case(c): the presence of surfactant SDS only, presence of MWCNT in 5% SDS, signal slightly effected. Excludes the direct reaction of MWCNT with cytochrome c that may occur. Figure.2

36. Conclusion Present data indicate that purified MWCNT are very effective scavengers of ROS, both (HO*) and (O2 *), no matter how such species are generated Inflammatory response elicited in rats by purified MWCNT is therefore ascribed to features other than particle generated radicals. MWCNT represent the first case of a potentially toxic particle which exhibits scavenging properties toward oxygen radicals, instead of generating them.

37. Further Research • The potential of purified single-wall or multiwall nanotubes to generate per se free radicals or ROS in acellular aqueous suspension has not been tested so far, further research should be implemented. • Detailed investigation and description of the precise mechanisms whereby MWNT can quench ROS should be investigated to fully understand the exact molecular mechanism taking place. Source: http://internetservices.readingeagle.com/blog/gavel/archives/microscope.jpg

38. In vitro evaluation of cytotoxicity of engineered carbon nanotubes in selected human cell lines XiaokeHu a, Sean Cook a, Peng Wang a, Huey-min Hwang a, Xi Liu b, Quinton L. Williams b *Note: All materials in the following section are obtained from the above research paper.

39. Introduction Due to their unique properties, carbon nanotubes (CNTs) have been used in various consumer, medical, and industrial applications Consequently, this expanding usage may lead to widespread human exposure via skin contact, ingestion, intravenous injection (in medical application) or by inhalation. Divergent literature reports of CNTs' toxicity make it difficult to conclude if any health risks are associated with CNTs exposure http://sites.uclouvain.be/pcpm/themes/TRANSPORT_NT_files/image001.jpg

40. Objective Systematic approach to study and compare the in vitro cytotoxicity of selected engineered carbon nanotubes (CNTs) to test cell lines including human skin keratinocytes, lung cells and lymphocytes http://www.babble.com/CS/blogs/strollerderby/2009/05/ivf.jpg

41. Materials and methods A3 lymphocyte cell line SWCNTs and MWCNTs prepared without further purification in distilled water to reach concentrations of 2, 5 and 10 ppm. A3 lymphocyte cell line Source: In vitro evaluation MSTO-211H lung cell MSTO-211H lung cell HaCaT human keratinocyte cell line Triad LT microplate reader measure the fluorescence of the samples in the FDA test Source : in vitro cytotoxicity study with human cancer cells.

42. Results Single Wall Carbon Nanotube (SWCNT) Results of fluorescein diacetate (FDA) uptake in T4-lymphocyte A3 cells indicated cytotoxicity caused by (SWCNTs) at 2, 5 and 10 ppm. At 2 ppm, the SWCNT treatment group retained 71.3% viability compared to the PBS control group. At 10 ppm, cellular viability further decreased to 56.5% of the PBS control group. In the skin keratinocyte HaCaT cells and lung MSTO-211H cells, the SWCNT did not demonstrate any cytotoxicity at concentrations of 2 and 5 ppm but slightly inhibited HaCaT cells and caused significant toxicity to MSTO -211H cells at 10 ppm.

43. Results (Cont.) Multi-Wall Carbon Nanotube (MWCNT) Multi-walled carbon nanotube (MWCNT) testing showed significant cytotoxicity to A3 cells in a dose dependent manner. At 10 ppm the viability of the cells decreased to 89.1% compared to the PBS control. In MSTO-211H cells, MWCNT caused significant toxicity at concentrations of 2 ppm and higher In HaCaT cells were inhibited by MWCNT significantly only at 10 ppm Figure 1 shows the normalized fluorescein diacetate uptake by A3 cells following 60-min treatment with 2, 5, and 10 ppm MWCNT Figure 1

44. Conclusions • Overall, the test CNTs inhibited cellular viabilities in a concentration, cell type, and CNT type-dependent pattern. The viabilities of the MWCNT-impacted cells are higher than the corresponding SWCNT groups. • The greater availability of defects and contaminants for cellular interaction may contribute to the higher cytotoxicity of SWCNT.

45. References • http://www.nanotech-now.com/nanotube-buckyball-sites.htm • “In vitro evaluation of cytotoxicity of engineered carbon nanotubes in selected human cell lines”. By XiaokeHu a, Sean Cook a, Peng Wang a, Huey-min Hwang a,⁎, Xi Liu b, Quinton L. Williams . • “Potential in vitro effects of carbon nanotubes on human aortic endothelial cells” by Valerie G. Walker a, Zheng Li a,1, Tracy Hulderman a, Diane Schwegler-Berry b,Michael L. Kashon c, Petia P. Simeonova a. a Toxicology and Molecular Biology Branch, Health Effects Laboratory Division. • “A Predictive Bayesian Dose-Response Assessment for Evaluating theToxicity of Carbon Nanotubes Relative to Crocidolite Using a Proposed Emergent Model”. By Jeffrey J. Iudicelloa;James D. Englehardt a • J.-P. Salvetat, J.-M. Bonard, N.H. Thomson, A.J. Kulik, L. Forro, W. Benoit, L. Zuppiroli. "Mechanical properties of carbon nanotubes." Appl. Phys. A (1999): 255–260.

46. References • Lam, Chiu-Wing, et al. "Pulmonary toxicity of Single-Wall Carbon Nanotubes in Mice 7 and 90 Days After Intratracheal Instillation." Toxicological Sciences (2004): 126-134. • Lu, Jian Ping. "Elastic Properties of Carbon Nanotubes and Nanoropes." Phys. Rev. Lett. (1997): 1297–1300. • Muller, J, F. Huaux and Lison D. "Respiratory toxicity of carbon nanotubes: How worried should we be?" Science Direct (2006): 1048-1056. • Ruoff, R. S. and D. C. Lorents. "Mechanical and thermal properties of carbon nanotubes." sciencedirect (1995): 925-930. • T. W. Ebbesen, H. J. Lezec, H. Hiura, J. W. Bennett, H. F. Ghaemi, T. Thio. "Electrical conductivity of individual carbon nanotubes." Nature (1996): 54-56.