NANO LETTERS
Biocompatibility and Toxicological Studies of Carbon Nanotubes Doped with Nitrogen
2006 Vol. 6, No. 8 1609-1616
J. C. Carrero-Sa´nchez,† A. L. Elı´as,‡ R. Mancilla,† G. Arrellı´n,† H. Terrones,‡ J. P. Laclette,*,† and M. Terrones*,‡ Instituto de InVestigaciones Biome´ dicas, UniVersidad Nacional Auto´ noma de Me´ xico, Cd. UniVersitaria, 04510, Me´ xico, D.F., and AdVanced Materials Department, IPICYT, Camino a la Presa San Jose´ 2055, San Luis Potosı´ 78216, Me´ xico Received March 10, 2006; Revised Manuscript Received June 7, 2006
ABSTRACT In this report, we compare the toxicological effects between pure carbon multiwalled nanotubes (MWNTs) and N-doped multiwalled carbon (CNx) nanotubes. Different doses of tubes were administered in various ways to mice: nasal, oral, intratracheal, and intraperitoneal. We have found that when MWNTs were injected into the mice’s trachea, the mice could die by dyspnea depending on the MWNTs doses. However, CNx nanotubes never caused the death of any mouse. We always found that CNx nanotubes were far more tolerated by the mice when compared to MWNTs. Extremely high concentrations of CNx nanotubes administrated directly into the mice’s trachea only induced granulomatous inflammatory responses. Importantly, all other routes of administration did not induce signs of distress or tissue changes on any treated mouse. We therefore believe that CNx nanotubes are less harmful than MWNTs or SWNTs and might be more advantageous for bioapplications.
Carbon nanoscience and technology has developed very rapidly over the past decade following the identification1,2 and bulk production3,4 of carbon nanotubes. Because of the remarkable mechanical, thermal, and electronic properties of carbon nanotubes,5 numerous applications have been conceived. For example, carbon nanotubes could be used as gas sensors,6,7 field-emission sources,8 polymer composite fillers,9 protein immobilizers,10 filters,11 electronic components,5,8 and so forth. However, a crucial issue, which needs to be addressed before nanotube-based components are commercially available, is toxicity. If nanotubes are used in novel products, then they are likely to get in contact with the human body through the skin and ocular surfaces and access the inner organs through the respiratory and digestive tracts. During manufacture, carbon nanotubes may enter the respiratory airways of the workers, and accumulate in the lungs. If nanotubes are used as fillers in food packaging products, then they could reach the stomach and intestines of the consumers. If cosmetics and bio-filters are developed using nanotubes, then they will certainly get in contact with human skin. Therefore, additional toxicological studies of these carbon nanomaterials need to be carried out. Previous reports have demonstrated that intratracheal injection of * To whom correspondence should be addressed. E-mail:
[email protected];
[email protected]. † Instituto de Investigaciones Biome ´ dicas, Universidad Nacional Auto´noma de Me´xico. ‡ Advanced Materials Department, IPICYT. 10.1021/nl060548p CCC: $33.50 Published on Web 07/07/2006
© 2006 American Chemical Society
single-walled carbon nanotubes (SWNTs) results in the death of the treated rats12 and mice,13 suggesting that they could be hazardous to humans. However, no information is available on the toxicity of multiwalled carbon nanotubes doped with nitrogen (CNx MWNTs); these tubes have demonstrated to have numerous potential applications.14-20 In this report, we compare the toxicological effects of two different types of carbon nanotubes, namely, MWNTs and N-doped multiwalled carbon (CNx) nanotubes. The tubes were administered in different ways to mice: nasal, oral, intratracheal, and intraperitoneal. We have found that MWNTs injected into the mouse’s trachea induce severe granulomatous inflammatory responses. Undoped MWNTs also caused the obstruction of small and medium-size bronchioles and damaged the bronchiolar wall that resulted in lung inflammation. However, CNx nanotubes were far more tolerated by the mice when compared to MWNTs. In particular, intratracheal injection of up to 5 mg/kg of CNx tubes neither caused the death of the mice nor induced visible signs of distress on the animals, in contrast to the lethal effects of similar doses of MWNTs or SWNTs12,13 administered to mice or rats through the same route. All other routes of administration of CNx and MWNTs did not induce signs of distress or tissue changes on the treated mice. We envisage that CNx nanotubes could be more biocompatible when compared to MWNTs or SWNTs and might be more advantageous for practical applications.
Figure 1. (a) Molecular model of a CNx nanotube indicating the two types of N groups attached on the tube surface, pyridine-type N, in which each N atom is bonded to two carbon atoms (see pink vertices), and subtitutional N, which corresponds to N atoms covalently liked to C atoms (see green vertices). (b) SEM image of a typical CNx nanotube carpet grown in our experiments showing tubes with average diameters of 30-50 nm and lengths of several micrometers (100-300 µm). (c) TEM image of CNx nanotubes showing the overall morphologies and compartmentalized structure of the tubes (bamboo-type). (d) HRTEM image of an individual CNx nanotube exhibiting bamboo-type compartments; see the arrow pointing to the edge of one compartment. The inset shows a higher magnification image of the graphitic planes of the CNx MWNT. (e) SEM image of pure carbon MWNTs. (f-g) HRTEM images of two different types of MWNTs with different diameters showing the excellent degree of crystallinity (note that the graphene planes are straight and parallel, see the inset in f).
The most common technique for producing bulk amounts of pure carbon nanotubes is based on the chemical vapor deposition (CVD) route, which consists on pyrolyzing organic molecules (e.g., CH4, C6H6, C2H2, etc) over a metal catalyst (e.g., Ni, Co, Fe) in an inert atmosphere.5 More recently, a new type of carbon nanotubes, known as CNx nanotubes, has been reported.14 These tubes are characterized by their bamboo-type morphology (Figure 1c and d) and contain N atoms embedded in the hexagonal carbon network at different ratios (1-10 at. %). Interestingly, the nitrogen is bonded to the carbon atoms in two fashions: (i) pyridinetype N, in which each N atom is bonded to two carbon atoms; this type of doping creates cavities within the side wall of the tube (Figure 1a), and (ii) substitutional N, which corresponds to N atoms bonded to three carbon atoms (Figure 1a). Because N contains an additional electron when compared to C, CNx nanotubes could exhibit metallic properties.15,16 Moreover, the N groups increase the reactivity on the graphene wall when compared to pure carbon nanotubes, which are basically inert. Because of this enhanced surface reactivity, CNx nanotubes have been used as fast responsive sensors,17 efficient and intense fieldemission sources 18, PS and epoxy composites,19 protein10 and nanoparticle immobilizers,20 and so forth. In addition, the incorporation of cyanide groups is plausible, and if so, then these tubes could be lethal. Therefore CNx nanotubes, which appear to be more reactive than pure MWNTs, may 1610
be hazardous and lethal, and therefore they need to be studied from the toxicological standpoint. We have carried out toxicological studies of CNx MWNTs and pure carbon MWNTs on mice. Several routes of administration were tested (nasal, oral, intratracheal, and intraperitoneal). In comparison with previous studies using SWNTs, our results show that CNx tubes appear to be far less harmful. For example, using extreme high doses of CNx nanotubes (e.g., 5 mg/kg), no lethal effects were observed on the mice, which is in contrast to previous reports using SWNTs.12,13 We have synthesized two types of multiwalled nanotubes: pure carbon MWCNTs and CNx MWNTs. We used the CVD process involving organic solvents as the carbon source in conjunction with ferrocene (FeCp2), which is used as a catalyst. Solutions containing 2.5 wt % of ferrocene in toluene (C7H8) were prepared and placed into a reservoir. The solution was then atomized using an abrupt Ar pressure difference.21 This generated spray (aerosol) was introduced into a quartz tube heated to 850 °C in the presence of Ar. Note that the SiOx tube served as the substrate for growing the nanotubes. This method has been described previously by Mayne et al.21 To produce CNx nanotubes (Figure 1bd), the toluene was exchanged by benzylamine (C7H9N).22 The nanotube growth proceeded for 15 min, using an argon flow of 3.7 L/min. The nitrogen content within these CNx Nano Lett., Vol. 6, No. 8, 2006
MWNTs has been determined using nuclear reaction analysis, and the samples treated contained ca. 2-4 wt % (not shown here). We should emphasize that higher nitrogen concentrations cannot be obtained using the experimental parameters indicated above. Both types of pristine nanotubes contained between 2 and 2.5 wt % of Fe (usually encapsulated in the tubes). Scanning electron microscopy (SEM) images of asproduced MWNTs and CNx MWNTs are shown in Figure 1b and e. At these magnifications, structural differences between the two samples are not evident. Even though MWNTs are usually longer than CNx MWNTs, their lengths are up to 450 µm (Figure 1e), whereas the CNx MWNTs’ maximum length only reaches the value of 300 µm (Figure 1b). High-resolution transmission electron microscopy (HRTEM) images exhibit the bamboo-like structure for CNx MWNTs (see the arrow in Figure 1d). For comparison, Figure 1f and g shows HRTEM images of undoped MWNTs. The degree of crystallinity within the graphene cylinders for MWNTs (undoped) is higher when compared to CNx MWNTs (Figure 1d, f, and g). In addition, we observe that CNx MWNTs possess a rougher surface than pure carbon MWNTs (see the insets from Figure 1d and f) caused by the presence of pyridinic nitrogen. In general, CNx tubes exhibit average diameters of 20-40 nm, whereas MWNTs could display slightly larger diameters (
