Biodistribution and clearance of instilled carbon nanotubes in rat lung
© Elgrabli et al; licensee BioMed Central Ltd. 2008
Received: 25 August 2008
Accepted: 09 December 2008
Published: 09 December 2008
Constituted only by carbon atoms, CNT are hydrophobic and hardly detectable in biological tissues. These properties make biokinetics and toxicology studies more complex.
We propose here a method to investigate the biopersistence of CNT in organism, based on detection of nickel, a metal present in the MWCNT we investigated.
Results and conclusion
Our results in rats that received MWCNT by intratracheal instillation, reveal that MWCNT can be eliminated and do not significantly cross the pulmonary barrier but are still present in lungs 6 months after a unique instillation. MWCNT structure was also showed to be chemically modified and cleaved in the lung. These results provide the first data of CNT biopersistence and clearance at 6 months after respiratory administration.
Given the low diameter of carbon nanotube (CNT), a possible translocation of this nanomaterial from the digestive tract to blood or from the lung to blood and then to other organs in the body must be considered. Inhaled ultrafine particles translocation has been previously described at low rate in a few studies with other particles like iridium or gold [1, 2]. Up to now, direct investigations of CNT translocation from the lung to the blood has not been conducted. The primary reason for that is the component structure of the CNT. Only made of carbon, CNT are difficult to distinguish from biological matrix. A few studies have used indirect methods to investigate CNT biokinetics (translocation, biodistribution, clearance...). Several biokinetics studies were performed by intra-venous injection of functionalized-CNT. Wang et al (2004) analyzed the biodistribution of SWCNT using a I125-SWCNT construct, and revealed indirectly by radioactivity measurement of I125, the presence of functionalized SWCNT in stomach, kidneys and bone. Similar observations were obtained in kidneys, liver and spleen by McDevitt et al (2007) with In111-SWCNT. Functionalized SWCNT were found in the different organs, and biokinetics studies showed a rapid clearance of the In111-SWCNT from blood and tissues [3–5]. One study followed the biokinetics of injected unmodified SWCNT using inherent near-infrared fluorescence of SWCNT for detection in blood and tissue samples . The authors report a rapid blood clearance and detected SWCNT only in the liver after 24 h. These results suggest a possible difference in biokinetics between modified and unmodified CNT. To investigate unmodified MWCNT biokinetics, Muller et al (2005) used another method based on the quantification of the CNT production metal catalyst residue . The production of carbon nanotubes by arc discharge , laser ablation  and chemical vapor deposition , use metal as catalysts such as iron, nickel, cobalt, etc...which always remain as impurities in the final product  and can be quantified. Here, we have first assessed the performances of using the metal catalyst of CNT as a tracer. We then analyzed unmodified Nickel-catalyzed MWCNT biokinetics after intratracheal instillation.
Analysis of metal impurities
Results showed the presence of 0.53% (w/w) of Ni, 0.08% (w/w) of S, 0.02% (w/w) of Mg, less than 0.01% (w/w) of Na and V and less than 0.005% (w/w) each for all other metals tested (listed in materials and methods).
Analysis of Ni-CNT bonds in the lung and in the lymph nods
Biodistribution of MWCNT
To validate our Ni dosage method, suspensions with known final CNT quantity (0, 6.25, 12.5, 25, 50 and 100 μg) were produced in quadruplicate. The MWCNT impurity study showed that Ni was present at an average concentration of 0.53% (w/w) of total MWCNT mass. Thus, the theoretical final Ni quantities in the control suspensions are respectively 0, 0.03, 0.06, 0.12, 0.25 and 0.5 μg. All prepared suspensions were mineralized and measured Ni quantities were determined by ICP-OES as it is described in materials and methods. No statistically significance differences between theoretical and measured Ni quantities were observed (data not shown) suggesting this dosage method is relevant to determine and quantify Ni. The detection limit for this dosage was determined to be 0.5 μg/L of Ni and correspond in our experimental conditions to 100 μg/L of MWCNT.
Quantification of Ni in rat's organs after instillation of MWCNT by intratracheal instillation.
Treatment time (days)
Lung after BAL
53 ± 12%
54 ± 10%
55 ± 8%
26 ± 6%
16 ± 9%
10 ± 2%
24 ± 5%
14 ± 2%
12 ± 2%
28 ± 2%
All other organs
About 50% of the instillated MWCNT were located in the parenchyma of the lung after 1 month. 10 to 24% of CNT were found in the alveolar cells during this period. At 30 days, 31% of total CNT injected was eliminated from the lung but 28% of this same nanotube was found in the lymph nods for a transition period less than 60 days. Clearance of MWCNT was amplified at 3 and 6 months after the exposure because 63% and 84% of total MWCNT injected was respectively eliminated from the organism (Table 1).
Clearance of MWCNT from the lung
Physical and chemical modification of MWCNT in vivo
After 15 days instillation of MWCNT in rat lung, chemical modifications of the MWCNT were assessed by infrared spectroscopy. Presence of alcohol, carbonyl and nitrogen function were observed on the MWCNT instillated to rat but not on the same suspension of MWCNT that was not injected in the presence or absence of cells residue (Figure 6D). Our results suggest the capacity of rat lung to chemically modify the MWCNT structure.
Biological lung response to MWCNT
Previous studies on MWCNT biodistribution were performed after tracer attachment on the surface of the tubes. But tracers modify MWCNT hydrophobicity and therefore MWCNT biodistribution. STEM-EDX analysis of our MWCNT reveals that Ni, which is an intrinsic impurity of the MWCNT we used, is strongly linked to the MWCNT and that MWCNT-Ni bonds are not affected in the body. Our results show that Ni can be used as native MWCNT tracer. More generally, utilization of metallic impurities as tracers can be a good approach to assess biokinetics of MWCNT in the body. However, the detection limit (here 0.01 μg/ml of Ni) does not allow the utilization of this method for very low concentration of MWCNT. In addition, the strength of the bond between the metal and the MWCNT can be different, depending on metals and CNT types. So that STEM-EDX bonding analysis should be performed for each CNT studied.
Ni quantification in several organs has revealed the presence of MWCNT only in the lungs and in the lymph nodes. 30 days after the instillation of MWCNT, the totality of particles was found in the lung (69%) and in the lymph nodes (28%). These results suggest no, or at least a negligible, elimination or biodistribution of CNT to other organs 1 month after instillation. Such an observation has already been done. In their study, Muller et al (2005) had found no nearly elimination of MWCNT in the rat lung 28 days after an intratracheal instillation. In our experiment, the presence of Ni in the lymph nodes was only observed at 30 days. This result can be explained by the absence of quantification between 7 days and 30 days and between 30 days and 90 days. Ni and so CNT is certainly not present in the lymph nods only 30 days after exposure but probably between more than 7 days and less than 90 days.
We have shown a decrease in the MWCNT quantity initially instilled 3 and 6 months after exposure. Three months after, only 37% of the MWCNT remained within the lungs and only 16% remained after 6 months. These observations are also in accordance with the results obtained by Muller et al, where only 18% of instilled intact MWCNT was eliminated in the lung after 60 days . The fact that we failed to detect MWCNT in various systemic organs is in favor of elimination rather than a translocation of MWCNT from the lungs. Nevertheless, due to the detection limit of our Ni dosage method, a very limited translocation (< 2% of MWCNT instillated) could not ruled out. This absence of alveolar passage was also described in vitro on pulmonary barrier . So, taken together, all these results show that MWCNT do not seem to significantly cross alveolar epithelial barrier and that they are eliminated from the lung. Previous studies on the elimination of man made mineral fibers (MMVF) showed a half clearance time near to 4 months . This result suggests that MWCNT can be eliminated from the lung with an equivalent time as others fibers in the lung.
The fact that the totality of instilled MWCNT was not detected at day 1 and day 7, (respectively 63% and 78%) is probably due to the experimental protocol. Indeed, MWCNT quantification in the BALF was performed after a centrifugation troughout a membrane as described in Materials and Methods section. At this step, free MWCNT that were present at 1 and 7 days were certainly eliminated and so was not quantified. This hypothesis is strengthened by the fact that difference observed between 1, 7 and 30 days is due to an augmentation of MWCNT in alveolar cells suggesting a diminution of free MWCNT part in the lung (Table 1).
Our study of MWCNT localization in the lung showed little modification of MWCNT quantity in the parenchyma, augmentation in alveolar cells (10% at day 1 and 24% at day 7) and absence in the BALF. In parallel, the observation of the alveolar cell population by optical microscopy reveals that the cells were mainly alveolar macrophages and that there was an increase in the phagocytosing cell number well correlated with the chemical dosage of MWCNT in the cellular part of the BALF. Engulfment of MWCNT by alveolar macrophages was also confirmed by TEM observations. This cell localization for MWCNT confirms the potential capacity of alveolar macrophages to phagocyte CNT as it was described before [16, 17].
In summary, we have shown in this work that metallic impurities of CNT may be used as tracers for CNT pharmacokinetics and toxicological studies, insofar as we verified the impurities-CNT bonds. This approach reveals that the MWCNT we used don't significantly cross the pulmonary barrier and can be eliminated. This elimination might to be due in part to macrophages. This hypothesis needs further investigations to be confirmed and to reach a better understanding of the fate of CNT in the body.
Materials and methods
MWCNT (product number: 636649) was purchased from Sigma -Aldrich (Lyon, France). These nanotubes were synthesized by Chimical Vapor Deposition method. Their diameter ranged from 20 to 50 nm and their length from 0.5 to 2 μm (data from the supplier).
Analysis of MWCNT metal impurities
MWCNT were mineralized in 5 ml nitric acid plus 5 ml chloridric acid at 180°C for 15 min in a microwave oven (MarsXpress, CEM, Matthews, NC). The mixture was filtrated and the volume was adjusted to 20 ml with distilled water. Samples were then analyzed for metal content (Li, Be, B, Na, Mg, Al, Si, S, K, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Ge, Se, Br, Rd, Sr, Y, Zr, Nb, Mo, Ru, Rh, Pd, Ag, Cd, In, Sn, Sb, Te, I, Cs, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hf, Ta, W, Re, Os, Ir, Pt, Au, hg, Tl, Pb, Bi, Th and U) by ICP-MS (Elan, 6100DRC, Perkin Elmer- Sciex).
Preparation of MWCNT suspensions
Different concentrations of MWCNT were suspended in NaCl in the presence of bovine serum albumin (BSA) at the same concentration as that of the MWCNT. The suspensions were sonicated 2 mn (5s pause every 10s) at 40 W with an ultrasonic probe (sonicator ultrasonic processor XL 2020, Misonix incorporated) as previously described . Using this dispersion method, more than 80% of total agglomerates have a size smaller than 10 μm and correspond to breathable agglomerates .
Male Sprague-Dawley rats weighing 180–220 g were purchased from Charles River Laboratories (St Germain-sur-l'Arbresle, France). The rats were kept in a conventional animal facility and had access ad libitum to food and drink. The experimental protocol has been approved by the local ethical committee for animal research.
Transmission Electron Microscopy (TEM) and CNT-Nickel bond analysis by Scanning and Energy Dispersive X ray microanalysis (STEM-EDX)
rats were exposed to NaCl-BSA as control and 6 rats were exposed to 2 mg of MWCNT by intratracheal instillation. This high quantity was chosen in order to facilitate the detection of CNT in the lung for STEM analysis. After 7 days, the animals were anesthetized. The lungs and the lymph nodes were removed, cut in small sections and fixed with 2.5% glutaraldehyde. After a postfixation with 1% osmium tetroxide, the samples were dehydrated by ethanol and embedded in EPON 812 (TAAB). The ultrathin sections of 90 nm and 150 nm respectively for TEM and STEM-EDX analysis were obtained by an ultramicrotome (UCT, Leica), mounted on copper grids and stained with uranyl acetate and examined in a Tecnai G2 Biotwin (FEI) electron microscope using an accelerating voltage of 100 kV. Several photographs of entire cells and of local detailed structures were taken, analysed and compared to NaCl-BSA control samples.
To analyze the bond of CNT and nickel in the body, STEM-EDX analyses were performed. The organ sections were prepared as described above for TEM, omitting the postfixation with osmium tetroxide to avoid an Os peak in the spectra. Unstained ultrathin sections were analysed with a dispersive X-ray microanalyser equipped with a Super Ultra Thin Window (SUTW) model SAPPHIR (EDAX), at 100 kV.
Intratracheal instillation studies
Rats were intratracheally instillated with 0, 1, 10 or 100 μg of MWCNT/rat and sacrified 1, 7, 30, 90 and 180 days later. For each concentration and for each recovery period, 2 groups of 6 rats (group A and group B) were used. Nanotube suspensions were prepared as previously described . Broncho alveolar lavage (BAL) and chemical quantification of Ni were performed on rats included in the group A. Histopathology and molecular biology were assessed on rats included in the group B.
Nickel dosage by Inductively Coupled Plasma -Optical Emission Spectrometer (ICP-OES)
Control and exposed animals of group A were anesthetized and the lungs, liver, kidneys, spleen, heart, brain, lymph nodes, thymus and testis were removed for ICP-OES nickel assay. The lung was separated in 3 compartments: the broncho alveolar lavage fluid (BALF) for detection of free MWCNT, the cellular part of the BAL for analysis the part of MWCNT engulfed in cells and the parenchyma of the lung. The organs were mineralized in 3.5 ml of nitric acid and 1.5 ml of chloridric acid for 4 h at 100°C and then treated with 1.5 ml of H2O2 for 1 h at 95°C on a heat bloc. The volume was adjusted to 20 ml with distilled water and samples were analyzed for nickel content by ICP-OES (Ultima, HORIBA Jobin Yvon, Edison, NJ).
BAL was performed on anesthetized animals included in the group A. The lungs were washed 3-times with 9 ml of phosphate buffered saline (PBS). The BAL was centrifuged (5 min, 15wa0 g at 4°C) and the cell-free BAL (BALF) was concentrated using centrifugation (2000 g, 4°C) in Amicon Ultra tubes® (Millipore) until the volume was reduced to 1 ml. The cell number was determined by counting with a Malassez cell. Differential cell count was performed on May-Grünwald-Giemsa-stained slides. Specific protein quantifications were performed in the concentrated BALF using bio-plex kit for IL10, GM-CSF (Biorad, Cat n°: 171K11070) according to the manufacturer's instructions.
Physical and chemical modification of MWCNT analysis by TEM and infrared spectroscopy
A 0.7 mg/ml suspension of MWCNT was produced as previously described. 150 μl of this suspension was instillated in 3 rats. After 15 days, rats were sacrified by intraperitoneal injection of pentobarbital and BAL were performed. A first centrifugation at 350 g for 10 min was performed to separate the MWCNT and the cells from the protein fraction. Cells lysis was then done in the presence of 300 μl of distillated water. After 1 h, 10 μl of this suspension was loaded on TEM copper grids to measure the length of MWCNT. In parallel, 2 types of control were realized. First control was prepared by loading directly 10 μl of the instillated suspension on TEM cooper grid. For the second control, BAL was performed on untreated rats. Cells were recovered by a 350 g centrifugation for 10 min. After removing the protein fraction, cells were lysed in the presence of water for 1 h and the suspension was put in presence of 0.7 mg of CNT. After 15 days at 37°C in oven, the suspension was centrifuged at 350 g for 10 min and resuspended in water at the same time of MWCNT instillated in rats. 10 μl of this suspension was loading on TEM cooper grid as control 2.
Treated MWCNT, control 1 and control 2 suspensions were then centrifuged at 350 g for 10 min. Supernatant was eliminated by pipeting and put at 37°C over night to obtain a dry powder. Chemical modification was then assessed by infrared spectroscopy (Nicolet 510M) on 70 μg of commercial MWCNT powder, control 1 powder, control 2 powder and treated MWCNT in the presence of 260 μg of KBr.
Phagocytosis makers' by reverse transcription real time polymerase chain reaction (RT-qPCR)
mg of lungs from group B rats was disrupted using Precellys® 24 lysis/homogenizer (Bertin technologies, France) and total RNA was extracted with RNeasy® plus Mini Kit according to manufacturer's protocol in presence of 600 μl of RLT buffer. 1 μg of total RNA was reverse transcribed to cDNA with Omniscript® Reverse Transcription kit (Qiagen, France) according to the manufacture's protocol using OligodT primer (Qiagen).
The quantification of mRNA for RPL32 (first housekeeping gene, Cat n° QT00444857), GNB2L1 (second housekeeping gene, Cat n° QT00193872), EEA1 (Cat n° QT01619121), β actin (Cat n° QT00193473), Elmo 1 (Cat n° QT01581930), Elmo 2 (Cat n° QT00365680), Elmo 3 (Cat n° QT00382214) and Dock 180 (Cat n° QT01684081), transcript were performed using QuantiTect™ Primer® Assay (Qiagen) and QuantiTect™ Sybr Green® Kit (Qiagen) according to the manufacturer's protocols on 100 ng of cDNA using Mastercycler® ep realplex 4 S (eppendorf, France). Each sample was run in triplicate.
All data were expressed as mean ± S.D (standard deviation). Differences between groups were assessed by the one-way analysis of variance (ANOVA). If the variances between groups were homogenous, groups were subjected to the multiple comparison Dunnett's test. If the variances were not homogeneous, groups were compared by the Mann-Whitney test. *P < 0.05 was considered as the statistical significance level.
This work has been supported by a grant of French Ministry for Ecology, Sustainable Development and Spatial Planning (MEDAD), BCRD05-DRC04 NANORIS TOXI (AP2005).
Jorge Boczkowski is supported by Inserm and Assistance Publique – Hôpitaux de Paris (Contrat d'Interface)".
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