BASIC SCIENCE

Nanomedicine: Nanotechnology, Biology, and Medicine9 (2013) 657–664

Original Article

Size-dependent biodistribution of carbon nanohorns in vivoMinfang Zhanga,⁎, Takashi Yamaguchib, Sumio Iijimaa,b, c, Masako Yudasakaa,⁎

aNanotube Research Center, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, JapanbInstitute for Advanced Research, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan

cDepartment of Materials Science and Engineering, Meijo University, Tenpaku, Nagoya, Japan

Received 12 July 2012; accepted 30 November 2012

nanomedjournal.com

Abstract

Carbon nanotubules, such as nanotubes and nanohorns, are potentially useful as drug delivery or hyperthermia agents for cancer therapy.However, the biokinetics of variously sized nanocarbons are important for their medical application and risk assessment. To examine thetime course of the biodistribution of carbon nanohorns (CNHs) in mice, CNH aggregates of 100 nm (L-CNHs) or CNHs of 30–50 nm(S-CNHs) were dispersed with lipid polyethylene glycol and administered to mice through tail vein injection. Histological observationrevealed that S-CNHs accumulated more slowly than did L-CNHs in the liver and spleen. The accumulation of L- and S-CNHs inspleen reached saturation within 1 and 48 h, respectively, and the accumulation in liver reached saturation within 48 h and N7 days,respectively. CNHs did not accumulate appreciably in the lung, skin, or kidney. Histologic, hematologic, and immunologic (IL-6, TNF-α, andIFN-γ) tests did not reveal obvious toxicologic lesions at any time point.

From the Clinical Editor: In this study the biodistribution and accumulation characteristics of small and large carbon nanohorns werecharacterized in mice. Data demonstrate slower accumulation of small carbon nanohorns in liver and spleen, no accumulation in skin, lung,or kidney, and no obvious hematologic or immunologic toxicity.© 2013 Elsevier Inc. All rights reserved.

Key words: Nanoparticle; Carbon nanohorns; Biodistribution; Biokinetics

Carbon nanotubules, such as nanotubes1,2 and nanohorns,3

have many potential medical functions, including tumor-target-ing drug delivery4–14 and imaging of diseased sites.15–18

Nanocarbons also have the intrinsic therapeutic effect of photo-hyperthermia, in which black nanocarbons absorb light anddissipate the optical energy as thermal energy. This releasedenergy heats the surrounding tissues7,13,19,20 and can be used inthe treatment of cancer. However, for carbon nanotubules to bepractically useful, their toxicity and biodistribution in the bodymust be fully understood. In this respect, almost all nanocarbontypes, including single-21–24 and multi-wall nanotubes25–27 andnanohorns,28,29 have been investigated. Although many para-meters, such as dose, particle size (long or short), and surfacefunctionalization, play key roles in nanocarbon toxicity andbiodistribution, the effects of these parameters are not fully under-stood. In particular, few studies have investigated the effect ofparticle size in biological systems in vivo.

⁎Corresponding authors:E-mail addresses: m-zhang@aist.go.jp (M. Zhang),

m-yudasaka@aist.go.jp (M. Yudasaka).

1549-9634/$ – see front matter © 2013 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.nano.2012.11.011

Please cite this article as: Zhang M, et al, Size-dependent biodistribution ofdx.doi.org/10.1016/j.nano.2012.11.011

In contrast to long, stiff multi-wall carbon nanotubes andbundles of single-wall carbon nanotubes, short-length nanotubes(i.e., of several hundred nanometers) and long, flexible carbonnanotubes have almost no toxicity in vivo. Typically, nanotubes≥10 μm in length and ≥100 nm in diameter elicit adverseeffects when injected into the peritoneal cavities of rodents.29 Thebiodistribution of nanocarbons administered in animals alsoseems to depend on their size. Intravenously injected carbonnanotubes tend to accumulate in livers and spleens. However,nanotubes ≤100 nm in length with properly functionalizedsurfaces have a long retention time in blood and can be partlyexcreted in the urine and feces.21,25 Therefore, small-sized carbonnanotubes might show improved stealth and excretion. However,the effects of size on the toxicity and biodistribution when carbonnanotubules are injected into the bloodstream are still unknown.Although the nanotube length can be shortened to 100–200 nmor less by strong acid treatment,30 fluorination,31 or surfactant-assisted ultrasonication,32,33 it is difficult to obtain a uniformnanotube length.

A carbon nanohorn (CNH) is a single-graphene tubule withhorn-shaped tip.3 Its diameter measures ~2 nm or less near the

carbon nanohorns in vivo. Nanomedicine: NBM 2013;9:657-664, http://

658 M. Zhang et al / Nanomedicine: Nanotechnology, Biology, and Medicine 9 (2013) 657–664

horn-shaped tip and ~5 nm in the tubule body, with an almostuniform length of about 30–50 nm. Nanohorns form sphericalaggregates with a diameter of ~100 nm, designated as large-sized CNHs (L-CNHs). Recently, individual CNHs withdynamic particle sizes of about 30–50 nm, so-called small-sized CNHs (S-CNHs), have been successfully separated fromtheir aggregates by ultrasonic exfoliation and sucrose densitygradient centrifugation.34 Fewer S- than L-CNHs are taken up bymacrophages or other cell lines in vitro,35 which suggests thatCNHs have size-dependent cellular uptakes similar to those ofgold or other types of nanoparticles.

In this study, we treated mice with CNHs of two sizes(S- and L-CNHs) via tail vein injection and investigated thesize effect of CNHs on toxicity and biodistribution from 1 hto 7 days by histological analysis and blood tests. The L- andS-CNHs showed size-dependent accumulation rates and lowtoxicity in the liver and spleen. Compared to L-CNHs, S-CNHsaccumulated more slowly in these organs, suggesting a longerblood circulation time and their potential utility as drugcarriers for targeted therapy. Because individual CNHs andsmall-sized aggregates have similar structures to single-wallcarbon nanotubes or their bundles, these size-effect results forCNHs may have implications for other nanocarbon/tubulemedical applications.

Figure 1. Results of transmission electron microscopy of L-CNHs (A) andS-CNHs (B) and dynamic light scattering of aqueous dispersions of S- andL-CNHs in LPEG/PBS (C).

Materials and methods

Preparation and structural analysis of L- and S-CNHs

To generate L-CNHs, the aggregates of CNHs, which wereproduced by CO2 laser ablation of graphite in Ar (ca. 760 Torr)without auxiliary heating,3 were oxidized by light-assistedoxidation, using hydrogen peroxide for enhancement of theirhydrophilic property.36 The obtained material (L-CNHs) wasdispersed in phosphate-buffered saline (PBS) by using lipidpolyethylene glycol (LPEG) (N-palmitoyl-sphingosine-1-{suc-cinyl-[methoxy(polyethylene glycol)5000]}, Avanti Polar lipidsInc.). The obtained mixture was treated by bath-type sonicationfor 10 min. The concentration of LPEG in PBS was 1 mg/mL,and the concentration of L-CNHs in LPEG/PBS solution wasabout 1 mg/mL.

To generate S-CNHs, the aggregates of CNHs (~100 nm)were oxidized in dry-air by heating them from roomtemperature to 450 °C at a rate of about 1 °C/min.37 Oxidizednanohorns were dispersed in an aqueous solution of LPEG(1 mg/mL) via sonication (horn-type sonicator) at 300 W forabout 1.5 h. After sonication, S-CNHs were separated fromaggregates by sucrose density gradient centrifugation.34 Theobtained S-CNHs were centrifuged with a filter-centrifuge tubeand washed with water five times to remove extra LPEG. TheS-CNHs were dispersed in a fresh solution of LPEG/PBS(1 mg/mL) via sonication (bath-type sonicator) for about10 min. The concentration of CNHs in LPEG/PBS solutionwas adjusted to ~1 mg/mL.

The S- and L-CNH structures were observed by transmis-sion electron microscopy (TEM; Topcon EM002B). Ramanspectra were measured with NRS2100 (JASCO) with anexcitation wavelength of 488 nm. The particle size distribu-

tions in the S- and L-CNH solutions were measured bydynamic light scattering (DLS, Photal Otsuka ElectronicsFPAR-1000).

In vivo biodistribution and toxicity analyses

Sixty 8-week-old male SIC:ICR mice were purchased fromSLC (Japan). All animals were maintained and bred in apathogen-free animal facility for 1 week. The animals weredivided into three groups: control, S-CNH, and L-CNH. A ~0.2-mL aliquot of S- or L-CNHs dispersed in LPEG/PBS or LPEG/PBS alone (control, 1 mg/mL) was injected into the tail vein ofmice (4.6 mg CNHs/kg body weight). Five mice were killed at 1,6, and 48 h and 7 days. About 2 mL of whole blood wascollected from the abdominal aorta. A full gross necropsy wasperformed for all animals. The liver, spleen, kidney, heart, lung,and skin were eviscerated, weighed, and fixed by 10% formalinneutral buffer solution. Organ parts were embedded in paraffinand cut into ~4-μm sections. Unstained and eosin-stainedsections were prepared and analyzed by optical microscopy. Weestimated the amount of L and S-CNHs in each of the tissues by

Figure 2. (A) Photographs of mouse spleens after intravenous injection of LPEG/PBS solution (control) and S- or L-CNHs LPEG/PBS dispersion. (B–E)Optical microscopic images of unstained mouse splenic tissues anatomized at 1 h (left) or 7 days (right) after single-dose intravenous administration of S-CNHs(B, C) or L-CNHs (D, E). Black spots in panels (B–E) are agglomerates of S- or L-CNHs.

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measuring of the areas occupied by black spots in about 20images (×40 magnification) using Winroof image analysissoftware (Mitani-visual, Japan).

Blood was collected in EDTA collection tubes (Becton,Dickson and Company, Japan). A 0.2-mL aliquot was used forthe hematology tests, which were performed with a bloodcorpuscle analyzer (XT-1800iV, Sysmex, Japan) to evaluate thenumber/quantity of red blood cells (RBC), white blood cells(WBC), platelets (PLT), neutrophils (NEUT), lymphocytes(LYMPH), monocytes (Mono), eosinophils (EO), and basophils(BASO), hemoglobin (HGB), hematocrit (HCT), mean corpuscu-

lar volume (MCV), and mean corpuscular hemoglobin (MCH).The remaining blood was centrifuged for cytokine analysis. Bloodplasma was prepared to quantify the concentrations of interleukin(IL)-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ,whichweremeasuredwith theQuantikinemouse IL-6 assay (R&DSystems), AssayMax mouse TNF-alpha ELISA kit (Assay Pro),and Mouse IFN-γ ELISA kit (Thermo Scientific), respectively.

All experiments were performed in compliance with the Japananimal protection laws and with approval by the institution'shuman or animal subject review committee of the NationalInstitute of Advanced Industrial Science and Technology.

Figure 3. (A) Photographs of mouse livers after intravenous injection of LPEG/PBS solution (control) and S- or L-CNHs LPEG/PBS dispersion. (B–E) Opticalmicroscopic images of unstained mouse liver tissues anatomized at 1 h (left) and 7 days (right) after single-dose intravenous administration of S-CNHs (B, C) orL-CNHs (D, E)). Black spots in panels (B–E) are agglomerates of S- or L-CNHs.

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Results

Structures of S-CNHs and L-CNHs

Figure 1, A and B shows typical TEM images of L- and S-CNHs, respectively. The L-CNHs comprised globular aggre-gates measuring about ~100 nm in diameter. The S-CNHsincluded individual CNHs (~80%) and small CNH aggregates(~20%; around ~50 nm in size). The individual CNHs were 30–50 nm in length and 2–6 nm in diameter. The number and size of

these particles were determined from TEM images (Supplemen-tal Information Figure SI-1). The Raman spectra showed that thelevel of structural defects in the S-CNHs was almost the same asthat in the L-CNHs (Supplemental information Figure SI-2).According to DLS, the particle size distributions of S- and L-CNHs dispersed in PBS with LPEG dispersant were 30–50 and100–120 nm, respectively (Figure 1, C). Dispersion in PBS washomogenous and stable over 1 month, consistent with a reportthat LPEG is one of the best dispersants for CNHs.38

Figure 4. Amounts of black spots of S- and L-CNHs in spleens (A) and livers(B) obtained by measurement of pigment areas from five plates. All data areexpressed as means±SD (n=5).

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Biodistribution

The S- and L-CNHs dispersed in PBS with LPEG wereintravenously injected into mouse tail veins at ~4.6 mg CNHs/kg body weight. Five mice were sacrificed at 1, 6, and 48 h and7 days postinjection. All animals survived the test periodswithout showing any signs of weakness or illness. No abnormalbody or organ weight changes were observed (SupportingInformation SI-3).

The spleens (Figure 2, A) and livers (Figure 3, A) becamedark in appearance after injection of S- and L-CNHs, withdarkening by S-CNHs occurring more slowly than that byL-CNHs. Darkening of other organs was not obvious (notshown). Typical optical microscopic images of the splenicand liver tissues at 1 h and 7 days are shown in Figures 2,B–E and 3, B–E, respectively. The area occupied by theblack spots, which represent agglomerates of S- or L-CNHs,was measured using image analysis software (Winroof). Datarepresent the average area from 20 images. The results showslower accumulation of S-CNHs compared to L-CNHs(Figure 4). The accumulation of L- and S-CNHs reachedsaturation in 1 and 48 h, respectively, in the spleen, and in48 h and N7 days, respectively, in the liver. The sloweraccumulation of S-CNHs suggests that their small size helpsto prevent their uptake by splenic and hepatic macrophages.

Further examination revealed that S- and L-CNHs accumu-lated at different sites in the spleen. The S-CNHs tended to bedistributed in red pulps, and their agglomeration sizes increasedwith time (Figure 5, A, Supporting Information, SI-4). At 1 h,the L-CNH agglomerates had large sizes, and most of them were

localized to the marginal zones. At later times, the L-CNHagglomerates were localized to the red pulps and marginal zones(Figure 5, B, Supporting Information, SI-5). In the liver,agglomerates of S- and L-CNHs were found in the hepaticsinusoids (Supporting Information, SI-6), where they weremostly taken up by Kupffer cells, in agreement with the resultsof a previous ultrastructural localization study.29

Despite the apparent accumulation of CNHs, histologicobservation revealed normal levels of inflammatory lesions,such as necrosis, cellular degeneration, and neutrophil infiltra-tion, in the livers and spleens (Supporting Information, SI-4, 5and 6). Obvious accumulation of S- and L-CNHs was notobserved in other organ sections of the lung, skin, or kidney,and toxicologic lesions were not found in these organs byhistologic observation.

Hematologic test results

The blood test results of the RBC, HGB, HCT, MCV andMCH did not show abnormalities caused by S- or L-CNHadministration (Supporting information SI-7). The WBC countswere in the normal range for mice given by S- or L-CNHs,39 butwere slightly lower than those of the control group (LPEG/PBS)at 1 h and 7 days (Figure 6, A). In addition, WBC differentialcounts, such as NEUT, LYMPH, Mono, EO, BASO, did notshow obvious systematic changes, indicating that no appreciableinflammation was caused by L- and S-CNHs (Figure 6, B–F).

Cytokines

Levels of proinflammatory cytokines (IL-6, IFN-γ, andTNF-α) in blood plasma were measured to evaluate possibleinflammatory reactions caused by S- and L-CNHs (Figure 6, G–I). The cytokine levels of the CNH-administered groups weresimilar to those of the control group. At 1 h postinjection, largequantities of TNF-α and IL-6 were observed in response to L-and S-CNH administration, respectively. Even in the controlgroup, large quantities of IFN-γ were seen. Thus, the largecytokine level at 1 h might reflect, in part, an initial acutereaction shortly after the intravenous injection. Because thecontrol, S-, and L-CNH groups showed similar cytokine levels atlater time points, it is unlikely that the S- and L-CNHs elicited theinflammatory response.

Discussion

Biodistribution

A few studies utilizing radiometal chelates as radiotracersobserved that functionalized nanotubes are not retained inorgans of the reticuloendothelial system (RES) but are clearedfrom the blood via renal excretion in 3 h after injection.25

However, aside from those findings, many studies show thatintravenously administered nanocarbons are mainly distributedin the liver and spleen of mice.21,23,26,29 Nevertheless, thebiokinetics of nanocarbon uptake by the RES system is notclear, especially at early time points (b24 h). Our data indicatethat nanocarbon accumulation in the liver and spleen is timedependent. Changes in CNH accumulation were size dependent;

Figure 5. Optical microscopic images with increasing magnifications of unstained mouse splenic tissues anatomized at 1 h (left) or 7 days (right) after single-dose intravenous administration of S-CNHs (A) or L-CNHs (B). Black spots of S-CNHs agglomerates tended to distribute in red pulps (RP), while the blackspots of L-CNHs agglomerates were large in size, and most were localized to the marginal zone (MZ). At 7 days, the agglomerates of L-CNHs were localized tothe red pulps (RP) and MZ. The agglomeration sizes of S-CNHs and L-CNHs increased with time. WP, white pulp.

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S-CNH accumulation in the spleen and liver took time longerthan 7 days, while L-CNH accumulation, reached saturation in48 h. These tendencies showed good agreement with theresults of previous in vitro tests,35 which show that L-CNHsare engulfed by macrophages more rapidly than S-CNHs.Usually macrophages rapidly take up particles in themicrometer size range, whereas the uptake of smaller particlesis slower.40 In this context, the higher uptake of L-CNH thanS-CNH seems reasonable.

To our knowledge, no study has examined the size-dependentbiodistribution of nanocarbons after their intravenous adminis-tration into animals. Some reports have investigated how sizeaffects the biodistribution of some none-carbon nanoparticles(e.g., gold,41–43 silicon,44 and polymeric nanoparticles45). Areport shows that the gold nanoparticles with sizes of 100 nm aremore accumulated in the liver and spleen than 50-nmnanoparticles at 24 h after injection.43 This is consistent withour results for S- and L-CNHs, and suggests that the differencein the biodistribution of 100- and 50-nm nanoparticles is notdependent on the material or surface conditions, but on the size.

Since the individual S-CNHs may be regarded as thickSWNTs, the S-CNHs accumulated in livers and spleen woulddecrease and be excreted in feces and urine if the animal testperiods were prolonged from 7 days to months like shortSWNTs (several hundred nanometer in length) reported by Liuet al.21 The smaller sizes (20–40 nm) of S-CNH might be more

superior to the larger size of L-CNH and longer length ofcarbon nanotubes (100 nm or longer) for the longer retentionperiod in the blood and a larger amount of excretion via urinaryor biliary routes.

Toxicity

The histological analysis, blood test, and cytokine measure-ment results showed that the L- and S-CNHs displayed lowtoxicity, which is consistent with our previous in vivoexperiments28 and other studies on nanotubes.24 The results ofhematologic tests performed in mice injected with S- and L-CNHs were almost the same as those in control mice. Miceinjected with L-CNH showed a slight decrease in the WBC counton Day 7, which may suggest an adverse effect on the immunesystem. This needs to be examined further in long-term animalstudies. Although the CNHs accumulated in macrophages in theliver and spleen, they did so with low toxicity, presumablybecause the graphene-based CNHs are chemically stable and notdegraded in macrophages. As a result, no antigen presentation bythe macrophages occurs28 and the inflammatory response isminimal. Additional consideration might need to be given to theinfluence of reactive oxygen species (ROS). A previous in vitrostudy showed that RAW264.7 macrophages secreted normalcytokine levels despite ROS up-regulation, which can lead to celldeath via lysosomal membrane destabilization.46 Another report

Figure 6. Hematology data of mice after intravenous injection of LPEG/PBS (control) or S- or L-CNHs dispersions. (A) White blood cell (WBC) numbers.WBC differential counts of neutrophils (NEUT%) (B), basophils (BASO) (C), monocytes (Mono) (D), eosinophils (EO) (E), and lymphocytes (LYMPH) (F).Concentrations of proinflammatory cytokines TNF-α, IL-6, and IFN-γ in blood plasma of mice after intravenous injection of LPEG/PBS (control) or S- orL-CNHs dispersions shown in panels (G), (H) and (I), respectively. *Pb0.05, **Pb0.01 vs. control groups. All data are expressed as means±SD (n=5).

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showed that CNH engulfment by primary macrophages resultedin an oxidative burst without a strong, concomitant increase inthe IL-6 secretion level.47 These results suggest that the ROSaction might be inhibited by CNHs in vivo or that ROS are usedfor the oxidation of CNHs. Previous observations point to theplausibility of this explanation. For example, carbon nanotubeshave been shown to be oxidized and degraded by ROS, such ashydroxyl radicals generated by the Fenton reaction.48,49

Conclusions

Our preliminary studies showed that small-sized S-CNHs(30–50 nm) accumulated in the spleen and liver more slowlythan large-sized aggregates of L-CNHs (~100 nm). In thespleen, S-CNHs were found in red pulps and the agglomerationsize increased with time, whereas L-CNHs were found at themarginal zones at all times and in red pulps at later times.Histologic, hematologic, and immunologic tests revealed that theCNHs had low toxicity.

Acknowledgments

We thank Drs. Azami and Kasuya (FundamentalResearch Laboratory, NEC) for the preparation of CNHs byCO2 laser ablation.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.nano.2012.11.011.

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