raman spectroscopy analysis and mapping the biodistribution of inhaled carbon nanotubes in the lungs...

Research Article

Received: 28 February 2012, Revised: 7 June 2012, Accepted: 7 June 2012 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jat.2796

Raman spectroscopy analysis and mapping thebiodistribution of inhaled carbon nanotubes inthe lungs and blood of miceTaylor Inglea, Enkeleda Dervishib, Alexandru R. Birisc, Thikra Mustafab,Roger A. Buchanana* and Alexandru S. Birisb*

ABSTRACT: Because of their small size, robust structure and unique characteristics, carbon nanotubes (CNTs) are increasinglybeing used in a variety of biomedical applications, materials and products. As their use increases, so does the probability of theirunintended release and human exposure. Therefore, it is important to establish their potential biodistribution and biopersistenceto better understand the potential effects of their exposure to humans. This study examines the distribution of CNTs in CD-1mice after exposure by inhalation of single-walled carbon nanotubes (SWCNTs) and investigates the possibility that inhalednanoparticles could enter the circulatory system via the lungs. Raman spectroscopy was employed for the detection of CNTsin lung tissue and blood based on their unique spectroscopic signatures. These studies have important implicationsconcerning the potential effects of exposure to SWCNTs and their use as potential transport vehicles in nanomedicine.Copyright © 2012 John Wiley & Sons, Ltd.

Keywords: Carbon nanotubes; Inhalation; Alveolar regions; Blood transport; Mice

Correspondence to: Roger A. Buchanan, Molecular BioScience Institute, Arkansastate University. 2713 Pawnee Street, Jonesboro, AR 72467. E-mail: [email protected]

lexandru S. Biris. Center for Integrative Nanotechnology Sciences, 2801 S. Universityve NANO Building Little Rock, AR 72204. E-mail: [email protected]

Molecular BioScience Institute, Arkansas State University

Center for Integrative Nanotechnology Sciences, University of Little Rock Arkansas

National Institute for Research and Development of Isotopic and MolecularTechnologies, Cluj-Napoca, Romania

Introduction

Owing to carbon nanotubes (CNTs) unique electronic, mechanical,optical and electrical properties (Dervishi et al., 2009a), single-walledcarbon nanotubes (SWCNTs) have found a variety of applications ina large number of technological areas, including nano-electronics(Baughman et al., 2002; Saini et al., 2011), energy harvestingand storage (Che et al., 1998; 1999) composite materials(Biswas et al., 2011; Harris, 1999; Treacy et al., 1996), sensing(Gooding, 2005; Pruneanu et al., 2011), medicine and biology(Biris et al., 2011; Karmakar et al., 2011, 2012; Li et al., 2007a,2007b; Mahmood et al., 2011).

The arrangement of carbon atoms in a hexagonal lattice tubewith a diameter of only a few nanometers provides a strong,chemically stable material with unique mechanical properties(Treacy et al., 1996). The continuous scientific interest that CNTshave produced has led to a plethora of large-scale applicationsresulting in the increased likelihood of unintended humancontact with these engineered nanomaterials. Human exposurecould potentially take place at various technological levelsincluding large-scale CNT production facilities, fabrication ofCNT-containing composite materials and various consumer pro-ducts (Dervishi et al., 2009a). As unique and promising as theseparticles are, their widespread use is still considered ’new andnovel‘, so there are rather few regulations or protocols to ensuresafety during their manufacturing, use, storage and disposal. Thestability of CNT structures combined with the lack or limitedenzymatic, digestive or biological processes capable of com-pletely degrading them into their constituent carbon atomsmake remediation after entry into the environment difficult. Alsoof great concern is existing data suggesting that their extremelylow one-dimensional characteristics and unique structure mayallow them to readily interact with various environmental and

J. Appl. Toxicol. 2012 Copyright © 2012 John

biological systems (Khodakovskaya et al., 2011; Mahmood et al.,2009). Thus, there is a real need for the development of complexand high resolution technologies that allow for rapid detection ofCNTs in environmental and biologically relevant substrates. Thebioavailability, uptake and distribution routes, accumulation ratesand fate of CNTs in the environment and the effects of humanexposure need to be well characterized. This is an area of intenseinvestigation and recently several research reports have beenpublished which were conducted to further understand CNTstoxicity (Zhang et al., 2011), fate and distribution in the environment(Khodakovskaya et al., 2011) and in living organisms (Karmakaret al., 2012; Liu et al., 2008; Wang et al., 2004).Given the fact that many of the methods for producing CNTs

involve extensive processing and chemical treatments, there isthe risk that these nanomaterials can become aerosolized andsubsequently inhaled by humans or animals. Therefore, it isextremely important to understand the pathways by which CNTsinteract with living organisms after inhalation. Several in vivostudies, in which CNTs were delivered into anesthetized miceby pharyngeal aspiration, have found toxicological responsesto exposure (Lam et al., 2006; Yu et al., 2008). Because this expo-sure technique allows precise amounts of CNTs to be delivered,

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it provides useful information about dose-responsive toxicology.However, it does not closely mimic the exposure routes, includinginhalation, that are most likely to be encountered by animals andhumans exposed to CNTs.

Of particular concern is that CNTs could lead to pathologicalpulmonary distress similar to that induced after exposure toanother high-aspect ratio particle, asbestos (Poland et al., 2008).The primary focus of Poland’s (2008) study was to examinethe relationship between the length of CNTs and asbestosfibers and their potential toxicity. Poland found that instillationof the described long CNTs (> 15 mm in length) was more likelyto induce inflammation and fibrosis within the peritoneal cavitythan exposure to asbestos fibers of equal lengths. It was notedthat, at equivalent doses, neither shorter fibers of asbestos norshorter CNTs produced significant inflammation. Toxicity inthese studies was attributed to the inability of the macro-phages to complete phagocytosis of the longer particleswhich inhibited their clearance from the peritoneal cavity. Thecontinued presence of the particles resulted in inflammation,granuloma formation and mesothelial cell damage. Poland’steam also established a pathogenicity paradigm to aid in theidentification of the potential toxicity of particles and identifiedthe geometry of CNTs as being a highly important characteris-tic of potential toxicity. They also reported that the composi-tion and biopersistence of CNTs could contribute to theirtoxicity (Poland et al., 2008).

To begin the process of defining the distribution of inhaledparticles, this study was designed to determine the pulmonarydeposition of inhaled SWCNTs in mice models. Animals in thesestudies were exposed to water aerosols containing dispersedSWCNTs to determine whether inhaled SWCNTs could infiltrateand accumulate within the lungs. Because non-functionalizedCNTs have been shown to rapidly aggregate, they are unlikelyto exist at the nanoscale in the air (Savetat et al., 1999). There-fore, the model we used did not seek to completely preventthe possible aggregation of CNTs after they were dispersed inwater. While this exposure technique does not allow precisemeasurements of the inhaled dose, it does allow deposition ofinhaled CNTs to be studied after exposure by a route likely tobe experienced by living organisms. Thus, this model is believedto be a good representation of a theoretical aerosol exposure.Moreover, we investigated the possibility that inhaled CNTscould reach alveolar compartments and translocate from thealveolar space into the bloodstream. Given the difficulty of iden-tifying the presence of CNTs in complex biological environ-ments, Raman spectroscopy was used to detect CNTs in thelungs and blood. This method is extremely sensitive to thestrong scattering characteristics of the CNTs, which makes itideal for resolving the presence of CNTs within biological tissues.Our studies clearly showed that inhaled SWCNTS were not onlydistributed throughout the lungs but were also in blood samplestaken from exposed animals, presumably because inhaled CNTscrossed the pulmonary epithelium and entered the bloodstream. Although the method of detection used and samplepreparation could not confirm that CNTs entered the alveoli,we surmise that they did because they were distributed withinthe lungs, and were not confined to the bronchi, bronchiolesor airways. It is also assumed that, as there was no observableevidence of damage to pulmonary tissue, SWCNTs enteredthe blood via alveolar capillaries. These results provide strongevidence that inhaled SWCNTs can readily penetrate deep intothe lungs and be transferred into the bloodstream resulting in

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potential exposure of all organs to SWCNTs. This raises thepossibility that, given the relative lack of knowledge about thelong-term fate of SWCNTs, their accumulation in tissues overtime, and the responses to such exposure, that inhaled SWCNTscould generate chronic toxic responses with undesired effects inhumans. These findings highlight the need to understand thecomplex interactions of CNTs with various biological systems bothin vitro and in vivo using a systematic approach to investigatetheir corresponding toxic effects at genetic, cellular and organismlevels, and to develop technologies for accurate and sensitive CNTdetection in tissues.

Materials and Methods

Single-Walled Carbon Nanotubes

Single-walled carbon nanotubes (SWCNTs) used in this experi-ment were synthesized using a Fe-Co/MgO catalytic systemwith a stoichiometric composition of 2.5:2.5:95 wt. % as previ-ously described by Dervishi et al. (2009b). Radio frequency(RF) catalytic chemical vapor deposition (cCVD) was themethod employed for the synthesis of the nanotubes, withmethane as the hydrocarbon source. For this, around 100mgof the powdered catalyst was spread into a thin uniform layeron the bottom of a graphite-made susceptor and which waspositioned in the center of a quartz tube reactor with an innerdiameter of 2.54 cm. The tube was then purged with a carriergas (in this case-argon) for over 10min at 150mlmin–1, in orderto remove the air present in the reactor, before the RF generator(which provides a very high heating rate of 300–350 �Cmin–1) wasactivated (Dervishi et al., 2009c).

Once the graphite boat temperature reached 850 �C, methanewas administered at a flow rate of 40mlmin–1 for 30min. Afterthe synthesis reaction ended, the system was cooled in thepresence of argon for another 10min. The as-produced SWCNTswere purified using diluted hydrochloric acid solution undergentle sonication.

The SWCNTs were characterized by electron microscopy,spectroscopy and thermal gravimetric analysis (TGA). Transmis-sion electron microscopy (TEM) images were collected using afield emission JEM-2100 F TEM [JEOL Inc. (Hirokazu Yamada 11Dearborn Rd.Peabody, MA 01960 USA)] equipped with a CCD cam-era. An acceleration voltage of 100 kV was used for analysis. For themicroscopy studies, SWCNTs were first dispersed homogeneouslyin 2-propanol under sonication for 30min. Next, several drops ofthe resulting suspension were placed on a TEM grid and dried un-der a vacuum (Dervishi et al., 2009c).

Raman scattering analysis was performed at room tempera-ture using a Horiba Jobin Yvon LabRam HR800 spectrometer(Edison, NJ) equipped with a CCD camera. The instrument isequipped with two gratings of 600 and 1800 lines mm–1. A HeNelaser (633 nm) was used as the excitation source, with the beamintensity measured at the sample surface of 5mW. The calibra-tion of the instrument was done with a silicon wafer at the521 cm-1 peak (Dervishi et al., 2007).

Thermogravimetric analyzes (TGA) to determine the thermalstability and purity of the carbon nanotubes were done undera dried air flow of 150mlmin–1 using a Mettler Toledo TGA/SDTA851e instrument (Columbus, OH). For this, approximately 3mgof the purified nanotube sample were heated in the thermal do-main of 25 to 850 �C with a rate of 5 �Cmin–1.

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Carbon nanotubes in lungs and blood as detected by Raman spectroscopy

Animals and Exposure

Adult CD-1 mice were housed in the Arkansas State University(ASU) PHS-assured animal facility, given food and water adlibitum and maintained on a 12/12 h light/dark cycle. All animaluse protocols were approved by the ASU IACUC. For acuteexposures, an alert, active animal was placed in a Plexi-glass™

box (volume ~2 liter) and exposed to a CNT-containing wateraerosol for 15 minutes. Unexposed animals (controls) weretreated identically except that the aerosol contained only waterand no CNTs. CNTs were sonicated in milli-Q water to producea uniform CNT suspension prior to exposure. A nebulizer[Nidek Pulmo-Mist (Birmingham, AL)] was used to produce anaerosol of the resulting CNT suspension. Next, 5ml of aerosol con-taining either 22ppm CNTs were introduced into the chamber dur-ing each exposure.

Tissue Collection

Animals were sacrificed 24 h after a single exposure. Animalswere anesthetized with isoflurane before sacrifice. Once theanimals were sacrificed, a jugular vein was severed and bloodwas collected with a pipette directly from the vein then trans-ferred into a microcentrifuge and immediately frozen in liquidN2. For lung collection, an incision was made along the anteriorside of the chest cavity to expose the lungs and trachea forremoval. The trachea was surgically exposed without puncturingthe pleural cavity and a ligature tied around the trachea tomaintain lung inflation. Immediately after removal, the lungand attached trachea were frozen in liquid N2. All samples werestored at �80 �C until analysis.

Sample Preparation

Frozen lungs were sliced into ~100 mM sections using a cryomi-crotome (Sakura Fineteck) at �20 �C. Slices were placed oncleaned glass slides, covered and dried at room temperaturebefore analysis. Digital images of each section were takenbefore drying to assist in determining the location where eachRaman spectrum was collected. Blood was thawed and trans-ferred onto cleaned glass slides, covered and dried at roomtemperature before analysis. Slides remained covered at roomtemperature until immediate analysis.

Figure 1. Transmission electron microscopy (TEM) images of the single-wal2009b) (Inset in Figure A), thermogravimetric analyzes (TGA) curve of purifieature (Dervishi et al., 2009c) (B) and Raman spectrum of the SWCNTs collecteet al., 2009c).


Data Processing and Statistics

Lab Spec Version 5.2324 was used to collect and process data.

Results and Discussions

Carbon Nanotube Analysis

The inset in Figure 1A shows the TEM image of bundles ofSWCNTs with uniform diameters between 0.8 and 2.4nm pro-duced using the RF-cCVD (Dervishi et al., 2009b). Thermogravi-metric analysis was used for characterizing the purity and thermalstability of the SWCNTs. Figure 1B shows the weight loss profilecurve of the SWCNTs synthesized by RF-cCVD; their quantitativeanalysis reveals that, after only one purification step, the nano-tubes have purity higher than 96% (Dervishi et al., 2009c). Theother curve in Fig. 1B presents the first derivative of the normal-ized TGA curve indicating that the nanotube combustion tem-perature is 570 �C (Dervishi et al., 2009c). It has been shownthat the thermal decomposition temperature of CNTs dependson their morphological properties; as the number of their gra-phitic walls increases, so does their combustion temperature(Arepalli et al., 2004; McKee and Vecchio, 2006). As previouslyreported by Dillon et al. (1999), purified DWCNTs decomposeat around 700 �C, which is much higher than the combustiontemperature of SWCNTs.Raman spectroscopy provided an understanding of the

crystalline properties and the diameter distribution of SWCNTs.The vibrational modes present in the as-collected Raman spectrawere the radial breathing mode (RBM), the D band, G band andthe 2D band (Dresselhaus et al., 2002; Kuzmany et al., 2001). As inmost of the cases, the nanotubes could be bundled togetherand this could affect the position of their RBM peaks whencompared with those of the isolated nanotubes. It was previ-ously shown through theoretical calculations, that for onenanotube, its diameter d, and the radial mode frequency oRBM

will be related through a relatively straightforward relationship:

oRBM cm�1� � ¼ a

d nmð Þ þ a;

where a= 234cm� 1 � nmand a=10 are constants that vary withthe excitation energy and the nanotubes bundle sizes (Raoet al., 2001). The D band, which is generally present between1305 and 1350 cm-1 is associated with the defects and other

led carbon nanotubes (SWCNTs) synthesized by RF-cCVD (Dervishi et al.,d CNTs, and the first derivative curve indicating the combustion temper-d with 633-nm laser excitation (A). RBM, radial breathing mode (Dervishi

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impurities in the graphitic structure of the CNTs. The tangentialband (or the G band), present between 1500 and 1605 cm-1, isassociated with the stretching modes of the carbon–carbonbonds in the graphene planar structure (Dervishi et al., 2009d).

Another significant mode that is commonly present in theRaman spectra for CNTs is the 2D band, or the second-orderharmonic of the D band. It is generally present between 2500and 2700 cm–1 and is highly dispersive and usually associatedwith the degree of nanotube crystallinity (Strong et al., 2003).The peak intensities for the D and G bands were recorded inorder to analyze the quality of the SWCNTs samples. Ramananalysis of the CNTs were performed before and after purifica-tion to make certain that the nanotubes remained structurallyundamaged after being exposed to mild hydrochloric acid (theacid was diluted in H2O at a ratio of 1:1).

Figure 1A shows the Raman spectrum of the CNTs used in thisstudy. These SWCNTs have an intense peak in the RBM region at190 cm–1 which corresponds to the nanotube diameter of 1.3nm(Dervishi et al., 2009c). Although RBM peaks give an indication ofthe CNT species present in the sample, they do not always providea very accurate indication of the actual relative percentage of thevarious nanotube diameters when analyzed with only one laser ex-citation (Dresselhaus et al., 2002). This occurs because Raman scat-tering for SWCNTs is believed to take place through a resonanceprocess that is related to the wavelength of the laser excitation(Rao et al., 2001). The G to D band intensity (IG/ID) taken from thenanotube Raman spectrum was calculated to be 8.2, indicatingrelatively highly crystalline CNTs. In addition, the D band presentin the Raman spectrum of these SWCNTs is very weak, and its linewidth is relatively small (35 cm-1) demonstrating the presence of avery small amount of amorphous carbon (Saito et al., 2005).

In vivo Carbon Nanotube Delivery by Inhalation

The biological impacts of exposure to CNTs are under extensiveinvestigation (Castranova, 2011), and several in vitro studies haveshown the ability of CNTs to enter tissues and cells (Muller et al.,2007, 2009). Yang et al. (2008) demonstrated that CNTs directlyinjected into the circulatory system were distributed to organswhere they induced inflammatory responses. In the describedexperiments, mice were injected with 40–1000mg of SWCNTsintravenously, and inflammation was assessed within the liver,lung and spleen 90 days after exposure (Yang et al., 2008). Liuet al. (2008) also found that the surface chemistry of theSWCNTs, referring to the added molecule for functionalization,affected the distribution and clearance. Liu et al. (2007) alsodemonstrated the ability of water-soluble, functionalizedSWCNTs to traverse the cell membrane after injection. TheseSWCNTs were found in the blood, liver, spleen, bone and skin.

There have also been a number of recent reports seeking toelucidate the mechanism underlying the toxicity of the CNTs.Alterations in reactive oxygen species (Azad et al., 2012; Pacurariet al., 2012), cell permeability, changes in enzymatic activity(Hitoshi et al., 2012) and activation of immunological responses(Qu et al., 2012) have been reported. Recently, CNT exposurehas also been linked with fibrogenic (He et al., 2011), cytotoxic(Cavallo et al., 2012), genotoxic (Lindberg et al., 2009) neurotoxic(Wei et al., 2011; Wu et al., 2005) and carcinogenic (Sargent et al.,2012) responses.

However, these studies report the effects of CNT exposureon cultured cells or after exposure by injection or pulmonaryintubation. While the information gained from such studies is


extremely important to the understanding of the potentialhealth impacts of exposure to CNTs, these reports do not reflectroutes by which exposure might actually occur. The small sizeof CNTs and their ability to be aerosolized makes inhalationa likely primary route of human exposure. In addition, severalphysiological processes could take place during inhalation thatmay not be fully captured by intubation exposure models. Whilethe effects of these features of the respiratory system on CNTbehavior have not been fully described, the behavior of othersmall particles as they are inhaled provides some relevant infor-mation about these impacts. For example, as they enter thehumid environment of the pharynx, larynx and trachea, CNTsmay agglomerate so that the actual size of the particles to whichthe organism is exposed may be much larger than the nanoscaleof isolated CNTs. CNTs will also undoubtedly interact with mucusand microvilli that line the entrance to the lungs. This may pre-vent a significant percentage of the inhaled CNTs from reachingthe pulmonary epithelium, but, owing to their extremely smallsize, may not completely prevent some inhaled CNTs from infil-trating the alveolar compartments.

The experimental design utilized in our experiments is shownin Fig. 2. The solubilized CNTs were delivered to the animalmodels by inhalation. Raman spectroscopy was used to charac-terize the deposition of CNTs in the deep lung tissues and todetermine if inhaled CNTs had entered the bloodstream.Becauseof the strong Raman scattering of the CNTs compared withbiological tissues and cells, this method is very powerful fordetecting and possibly quantifying the CNTs in various biologi-cal systems, including cells, organs, blood and the lymphaticsystem (Biris et al., 2009; Liu et al., 2008). Raman spectroscopyalso has the advantage of being able to detect agglomeratedas well as isolated CNTs. As CNTs are likely to agglomeraterapidly in biological matrices, this process could actually help intheir detection given the more enhanced Raman signal providedby the clustered compared with individual nanotubes. We havepreviously demonstrated the ability of Raman spectroscopy tovery accurately detect CNTs in blood and lymph circulation at veryshort acquisition times (Biris et al., 2009). The unique features ofCNTs’ Raman spectra, integrated with their strong optical scattering,allowed us to also detect CNT-tagged single cancer cells incirculation, indicating the specificity of this technique for theaccurate detection of CNTs in complex biological systems.Moreover, the 2D mapping capacity of this technique couldplay a further role in providing a method for the understandingof the bio-distribution of CNTs in vivo.

Among the CNT characteristic Raman bands, the G band has thehighest intensity and therefore is the most useful for identifyingCNTs in biological tissues and providing information on theirrelative concentration in the focal volume of the laser. A represen-tative Raman spectrum from a thin section (~100mm thick) of lungtissue from control and the animals that inhaled CNTs as describedabove is shown in Fig. 3.

The Raman analysis shown in Fig. 3 clearly indicates that theCNTs have reached the alveolar regions of the lung. Moreover,we found the nanotubes to be clustered and the representativespectra presented in Fig. 3B were collected from the spots thatprovided CNT-specific signal indicating the presence of thesenanomaterials. While nanoparticles within the pulmonary com-partment may have significant health impacts, information aboutthe effectiveness of the pulmonary epithelial barrier in preventinginhaled CNTs from entering the bloodstream could play a role inmitigating the effects of exposure. Thus, we conducted a series


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Figure 2. Experimental schematic utilized for the study of carbon nanotube (CNT) inhalation and possible transfer into the bloodstream.


of analyzes to determine if inhaled CNTs cross the pulmonaryepithelium and enter the bloodstream. If the inhaled CNTs crossthe epithelial barrier and enter the bloodstream, they would becarried to all parts of the body and could exert effects that extendfar beyond the pulmonary system. On the other hand, afterreaching the blood stream, inhaled CNTs could be rapidly cleared

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Figure 3. Raman analysis of the thin lung sections collected from the anim500–2000 cm-1) (A). Lung samples prepared by sectioning the tissue into tspectral range between 1500 and 1650 cm-1 of the lung tissues collectedexposed to CNTs by inhalation of 20 ppb or 22 ppm; samples were colleccomponents (spectral range of 1500–1650 cm-1) clearly indicate the presein the tissues harvested from the animals that were exposed to the CNTnanotubes (black arrow). 2D Raman mapping was performed on slices of lunand the intensity of the CNT’s G band was mapped as a function of the X–represent the CNTs) is clearly seen, as highlighted by the Raman mapping


from the body by excretion via the intestine, kidneys and skin, thussignificantly reducing the likelihood of deleterious effects onhealth. Therefore, blood samples were collected from the animals24 h after their CNT exposure by inhalation.The blood samples were analyzed by Raman spectroscopy,

and again the G band was used to detect the presence of

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als that were not exposed to carbon nanotubes (CNTs) (spectral rangehin layers were used for the Raman analysis (B); Raman analysis of thefrom animals that were not exposed to CNTs (control) and animalsted 24 h post exposure (C). Data deconvolution into Lorentzian peaknce of an additional band at around 1584 cm-1, which is only presents by inhalation. This extra band is associated with the G band of theg collected from the animals exposed to CNTs (D) with ~1 mm step size,Y axis. A wide distribution of the CNTs in the lung tissues (black spotsanalysis (D).


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SWCNTs in the blood. The spectra collected from blood samplestaken from the animals that had inhaled SWCNTs, presented inFig. 4, showed that, in several analysis points, a low intensityRaman peak (with a morphology of a shoulder over the1575 cm-1 peak) at around 1584 cm-1 was detected, which wasnot detected in the samples taken from animals that had notinhaled SWCNTs. Mapping of the blood film indicated that thispeak has a low intensity and was only detected in several spotsalong the blood films. This finding indicates that inhaledSWCNTs reached the blood stream, strongly suggesting thatthey had crossed the pulmonary epithelium. These results alsoshow that, 24 h after the exposure, they were present in theblood samples in large enough quantities to be detected byRaman spectroscopy. Further studies need to be performed todetermine the kinetics of the concentration distribution on theblood and their induced possible toxic effects. Furthermore, thisanalysis suggests that the CNTs are present within the bloodsamples, and we found clusters with an average size of severalmicrometers (Fig. 5). To prove the size of the CNT clusters inthe blood sample, we scanned linearly with a step size of 1mm.The results are shown in Fig. 5 and we found that the SWCNTscluster with sizes (in one direction) in the range of micrometers.The deconvolution into individual Lorentzian curves, for thespectra collected at these two points (Points 3 and 4) indicatedthe presence of the 1584 cm-1 peak, corresponding to thepresence of CNTs. It is also possible that smaller CNT clusters

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Figure 4. Raman analysis of the blood samples collected from the control an650–1800 cm-1) (A). Inset represents the scanning electron microscopy (SEMspectra, collected in the spectral range of 1500 to 1650 cm-1, for blood sampleand animals exposed to CNTs by inhalation (black line) (B). This analysis cleawith the G band of the CNTs, and is only present in the blood samples collecsamples were collected 24 h post CNT exposure of ~22 ppm suspension. Thezian curves (C). It can be clearly observed that the presence of a 1584 cm-1 p


(even individual SWCNTs) could be present in the samples, buttheir corresponding Raman G band intensity was below thedetection threshold of the spectrometer. Also given the fact thatwe did monitor in only a limited number of points, we cannotexclude the presence of CNT clusters of various dimensions.Actually, our analysis indicates a significant variation in the con-centration of the nanotubes detected in various animals in thelungs and blood. Especially for the blood samples, the detectionof the CNTs was rather difficult and large variations in thepresence of CNTs from one animal to another were observed.Given the limitations of Raman spectroscopy it is rather difficultto quantify the amount of nanotubes present in various tissues,but this method proved to be sensitive enough for the detectionof these nanomaterials in complex tissues. This highlights thecomplex clearing mechanisms that take place for CNTs and theirvariation from one organism to another one and are in line withprevious reports (Kreyling et al. 2009; Nemmar et al., 2002).

In light of the possible toxic effects of SWCNTs, these findingshighlight the need to further understand the interactions betweenCNTs and living organisms (Clichici et al., 2012). Inhalation wasfound to be a viable route of entry. Because the inhaled SWCNTscrossed the pulmonary epithelium and avoided pulmonarydefenses, they were dispersed throughout the circulatory system.We have shown here that inhaled SWCNTs, which tend to aggre-gate in biological media, can transition through the airways,alveolar walls, and pulmonary interstitium to reach the blood

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imals that were not exposed to carbon nanotubes (CNTs) (spectral range) of a blood sample, which shows the presence of blood cells. Ramans collected from animals that were not exposed to CNTs (control-red line)rly indicates the presence of an additional band at 1584 cm-1, associatedted from animals that were exposed to the CNTs (black arrow). The blood1550–1650 cm-1 spectra range was deconvoluted into individual Lorent-eak only occurs for the samples exposed to CNTs by inhalation.


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Point 3 Point 4

Figure 5. Linear scanning Raman analysis determining the one-dimensional size of a carbon nanotubes (CNT) cluster. For this study, the laser beamwas moved at 1 mm step sizes along the blood film and the resulting spectra were analyzed for the presence of the CNT-specific G band. The spectrawere deconvoluted in Lorentzian curves and only Points 3 and 4 indicated the presence of a 1584 cm-1 peak and the corresponding areas under thepeak were used for representation. The laser power of the 633-nm laser was 5mW at the surface of the sample.


stream, which could therefore bio-distribute them to variousorgans. This may enhance their utility as delivery vehicles fordrugs or active ingredients – not only for the treatment oflung-specific conditions and diseases, such as cancer, emphysema,or inflammation, but also for systemic treatments or treatmentstargeted to other organs. Previous research has shown thatfunctionalized SWCNTs can enter cells via endocytosis andintrinsic interactions between the particle and the lipid bilayer(Liu et al., 2007; Shi et al., 2008) making CNTs a potential vehiclefor transporting molecules into cells. It has also been shownthat CNTs can enter cells with larger attachments, such asplasmids (Liu et al., 2007) proteins and short interfering RNAs(Kam and Dai, 2005), Kam et al., 2005. This suggests that CNTscoupled with rather large molecules can translocate into indi-vidual cells allowing for a more targeted and direct transportof molecules into specific targeted cell populations withoutexposing untargeted cells. The results of this study showed thatSWCNTs reach the deep alveolar regions of the lungs after inha-lation and are subsequently transported into the blood stream,not only highlighting their extremely high mobility in crossingmembranes, but also suggesting that they can be deliveredby inhalation to other tissues and organs. Further studies arerequired in order to be able to fully quantify the nanotube accu-mulation in lungs and their bio-distribution to various organsvia the bloodstream or lymphatic system.

ConclusionsThis study has shown that untreated SWCNTs inhaled aftersuspension in a water aerosol enter the pulmonary system andcan be distributed throughout the body. The detection ofSWCNTs within the lungs shows that, once the particles areinhaled, they can clear the larynx and trachea and penetratedeep into the lungs. Detection of SWCNTs in the blood ofexposed animals shows that the inhaled SWCNTs crossed the


pulmonary epithelium and entered the bloodstream. The abilityof SWCNTS to enter the circulatory system heightens the potentialfor exposure of other systems and organs. It is unclear whetherthe particles would bioaccumulate or be excreted. Ramanspectroscopy was shown to be very useful in successfully detectingSWCNT presence in the lung tissue and blood of mice.Thus, these results may have important implications concerning

the potentially harmful and/or beneficial effects of nanoparticlesand provide a basis for further investigations targeting specificeffects of inhaled nanoparticles. They also raise the interestingpossibility of using inhaled nanoparticles for rapid delivery ofsystemic and targeted therapeutics.

Acknowledgments

Financial support from Arkansas Science and TechnologyAuthority grant no. 08-CAT-03; the National Center for ResearchResources (5P20RR016460.11) and the US Army Telemedicineand Advanced Research Center are highly appreciated.

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