Nanomedicine: Nanotechnology, Biology, and Medicinexx (2014) xxx–xxx

nanomedjournal.com

In vivo biodistribution of platinum-based drugs encapsulated intomulti-walled carbon nanotubes

Jian Li, PhDa, Aakansha Panta, Chee Fei Chinb, Wee Han Ang, PhDb,⁎,Cécilia Ménard-Moyon, PhDc, Tapas R. Nayak, PhDa, Dan Gibson, PhDd,

Sundara Ramaprabhu, PhDe, Tomasz Panczyk, PhDf, Alberto Bianco, PhDc,⁎,Giorgia Pastorin, PhDa,g,h,⁎

aDepartment of Pharmacy, National University of Singapore, Science Drive 2, SingaporebDepartment of Chemistry, National University of Singapore, Singapore

cCNRS, Institut de Biologie Moléculaire et Cellulaire, Laboratoire d’Immunopathologie et Chimie Thérapeutique, Strasbourg, FrancedSchool of Pharmacy, The Hebrew University of Jerusalem, Israel

eDepartment of Physics, Indian Institute of Technology Madras, Chennai 600 036, IndiafInstitute of Catalysis and Surface Chemistry, Polish Academy of Sciences ul. Niezapominajek 8, 30239 Cracow, Poland

gNUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences (CeLS), 28 Medical Drive, SingaporehNUSNNI-NanoCore, National University of Singapore, T-Lab Level 11, 5A Engineering Drive 1, Singapore

Received 28 August 2013; accepted 17 January 2014

Abstract

Carbon nanotubes (CNTs) are promising drug delivery systems due to their external functionalizable surface and their hollowed cavitythat can encapsulate several bioactive molecules. In this study, the chemotherapeutic drug cisplatin or an inert platinum(IV) complex wereentrapped inside functionalized-multi-walled-CNTs and intravenously injected into mice to investigate the influence of CNTs on thebiodistribution of Pt-based molecules. The platinum levels in vital organs suggested that functionalized-CNTs did not affect cisplatindistribution, while they significantly enhanced the accumulation of Pt(IV) sample in some tissues (e.g. in the lungs, suggesting their potentialapplication in lung cancer therapy) and reduced both kidney and liver accumulation (thus decreasing eventual nephrotoxicity, a typical sideeffect of cisplatin). Concurrently, CNTs did not induce any intrinsic abnormal immune response or inflammation, as confirmed by normalcytokine levels and histological evaluations. Therefore, functionalized nanotubes represent an efficient nano-carrier to improve accumulationof Pt species in targeted tissues/organs.© 2014 Elsevier Inc. All rights reserved.

Key words: Carbon nanotubes; Platinum-based drugs; Biodistribution; Drug delivery; Histopathology

This research has been supported by the National University ofSingapore, Department of Pharmacy [(AcRF) Tier 1-FRC Grant R-148-000-164-112] and by MOE of Singapore (GrantMOE2009-T2-2-011, R-398-000-068-112). The authors G.P. and W.H. A. acknowledge support also byA*STAR SERC TSRP-Integrated Nano-Photo-Bio Interface grant (ProjectNumber: 102152 0016). G.P. and A.B. acknowledge the support from the2011 Merlion Programme (project number: R-148-000-162-133).

⁎Corresponding authors.E-mail addresses: chmawh@nus.edu.sg (W.H. Ang),

a.bianco@ibmc-cnrs.unistra.fr (A. Bianco), phapg@nus.edu.sg (G. Pastorin).

http://dx.doi.org/10.1016/j.nano.2014.01.0041549-9634/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Li J., et al., In vivo biodistribution of platinum-baseNBM 2014;xx:1-11, http://dx.doi.org/10.1016/j.nano.2014.01.004

Background

Platinum-based drugs have been extensively used in mono-therapies or combination therapies for numerous solid malig-nancies. Cisplatin (Platinol®, CDDP), carboplatin (Paraplatin®),and oxaliplatin (Eloxatin®) are three main platinum drugsapproved by FDA and applied to clinical therapies for ovarian,1,2

lung,3,4 testicular,5 colorectal,6 and gastric cancers,7 and forlymphomas.8 Platinum drugs induce DNA interstrand cross-links that interfere with DNA, which finally lead to apoptotic celldeath involving prolonged cell cycle (G2) arrest.

9

However, a major concern about the therapeutic efficacy isthe biodistribution of platinum drugs, which may limit their

d drugs encapsulated into multi-walled carbon nanotubes. Nanomedicine:

Figure 1. Structure of the prodrug Pt(IV).

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accumulation in diseased tissues and induce toxicity in normaltissues. In previous studies on intraperitoneal (i.p.) injection ofCDDP, platinum concentrations in liver and kidneys weremarkedly higher than in lungs and plasma during a 21 daypharmacokinetic study.10 Apart from Pt(II) analogues, Pt(IV)complexes, with two additional coordination sites, weresynthesized as prodrugs to be strategically converted to theiractive Pt(II) form upon the cleavage of the axial ligands. Thebiodistribution study of Pt(IV) derivatives also revealed thatplatinum species mostly accumulated in kidneys and liver.11,12

Furthermore, it has been demonstrated that platinum drugs havesome side effects: nausea, nephrotoxicity, ototoxicity, neurotox-icity, bone marrow suppression, thrombocytopenia and periph-eral neuropathy are common with Pt-based drugs.13–18 Takinginto account the distribution propensity of platinum-based drugsin some specific organs and tissues, these adverse effects mayaggravate damage to normal organs, especially kidneys and liver,which have higher platinum concentrations in comparison withother tissues.19 Therefore, efforts to alter the biodistribution ofplatinum-based drugs are necessary to enhance their antineo-plastic effects and reduce side effects.

Towards that purpose, in the present study we performed theentrapment of platinum-based compounds within pristine andmodified multi-walled carbon nanotubes (MWCNTs) as drugdelivery systems. After administration in mice, we examinedthe tissue distribution of platinum into various organs. Thus far,a number of biodistribution studies on pristine or functionalizedCNTs have been published, showing contrasting effects ofCNTs onto the reticuloendothelial system (RES) in liver andspleen.20–30

In our investigation, we carried out a study on the influence ofthe CNT surface functionalization on the biodistribution in vivoof platinum-based species. The drug prototypes [namely CDDPand Pt(IV) prodrug] were entrapped within different MWCNTswhich, in view of their larger diameter in comparison withSWCNTs, provide the advantage of encapsulating a higheramount of drug molecules.

Materials and methods

Chemicals and reagents

Tetramethylammonium hydroxide (TMAH), 25% w/w aque-ous solution was purchased from Alfa Aesar. cis-Diamminepla-tinum(II) dichloride (cisplatin), ultrapure nitric acid (≥65%, forultratrace analysis), Platinum Standard for ICP (1000 mg/L Pt inhydrochloric acid), formaldehyde solution (36.5–38% in H2O),and 2,2′-(ethylenedioxy)diethylamine were purchased fromSigma Aldrich. Mouse IL-1β, IL-6, TNF-α ELISA ‘Ready-SET-Go!’ kits were purchased from eBioscience. cis,cis,trans-Pt(NH3)2Cl2(O-C(CO)–C6H5)2 (compound 1, Figure 1) wassynthesized in the Laboratory of Dr. Ang (National Universityof Singapore). Ultrapure MWCNTs were provided by Prof.Ramaprabhu (Indian Institute of Technology, Chennai, India):the outer and inner diameters of ultrapure MWCNTs (purityN95%) are 30–40 nm and approximately 10 nm, respectively;their length ranged from 200 nanometers to several microns.

Instruments

TEM was carried out using JEM 2010F HR-TEM (JEOL).UV–vis absorbance was measured using SHIMADZU UV-1800Spectrophotometer. Inductively coupled plasma opticalemission spectrometry (ICP-MS) was carried out usingAgilent 7500A inductively coupled plasma mass spectrom-eter. Thermogravimetric analysis (TGA) using TGA-SDT2960 Simultaneous DTA-TGA and Inductively CoupledPlasma Optical Emission Spectrometry (ICP-OES) usingPerkin ElmeDual-view Optima 5300 DV Inductively CoupledPlasma Optical Emission Spectrometer were performed byCMMAC (NUS).

Animals

Female BALB/c mice, 6–8 weeks old and weighing 18–22 g,were obtained from Center for Animal Resources (CARE) inComparative Medicine, National University of Singapore(NUS). The animals were housed in plastic cages (5 mice/cage) and allowed free access to sterile food pellets and water.The animal holding room kept a 12 h light/dark cycle. All animalstudies (protocol number IACUC 063/11) were performed incompliance with guidelines set by NUS Institutional AnimalCare and Use Committee (IACUC).

Synthesis of MWCNTOX and MWCNTTEG

Ultrapure pristine MWCNTs (10 mg) were oxidized byultrasonication in the presence of acidic mixture (4 mL)comprising 95% sulfuric acid and 65% nitric acid (3:1 v/v) for6 hours. The acid-treated MWCNTs were diluted with deionizedwater and filtered through 0.2 μm hydrophilic PTFE membrane.The residue was washed exhaustively with deionized water untilit turned into neutral pH and then dried in vacuum to yieldoxidized MWCNTs (MWCNTOX).

As-prepared MWCNTOX (9.7 mg) were dispersed in 1 mL ofdimethylformamide (DMF) using bath sonication, and then NHS(45 μmol), EDC·HCl (112 μmol), 2,2′-(ethylenedioxy)diethyla-mine (TEG, 111 μmol), and N,N-diisopropylethylamine(DIPEA) (112 μmol) were added in the suspension (Scheme 1in SI). The mixture was heated at 60°C for 24 h and filtered

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through a 0.22 μm PTFE membrane. The residue was washedwith DMF and deionized water, dried in vacuum to yieldMWCNTTEG. The degree of amino-functionalization per gram ofMWCNTTEG (amino-loading) was calculated through quantita-tive Kaiser test.

Entrapment of CDDP in pristine MWCNTs, MWCNTOX andMWCNTTEG via nano-extraction

The entrapment of CDDP in pristine MWCNTs has beenreported previously.31 The same procedure was carried out toprepare CDDP@MWCNTOX and CDDP@MWCNTTEG com-plexes. In brief, CDDP (7.5 mg) was mixed with MWCNTs (3.0mg) in ethyl acetate (EA, 1.5 mL) for 24 h and filtered through ahydrophobic PTFE membrane. The residue was washedextensively with a mixture (2.6:1:1 v/v EA:EtOH:water) anddried in vacuum to yield CDDP@MWCNTOX or CDDP@MWCNTTEG. Platinum content was determined using ICP-OESafter combustion by TGA. The samples were heated in air at arate of 10°C/min until a final temperature of 1000°C wasreached. The residue remaining after TGA was dissolved in aquaregia and diluted with 2% HNO3 for ICP-OES determination ofplatinum levels.

Entrapment of compound 1 in MWCNTOX and MWCNTTEG vianano-extraction

Our previous study has described the method for entrapmentof Pt(IV) compound 1 into pristine MWCNTs.32 The preparationof Pt(IV)@MWCNTOX and Pt(IV)@MWCNTTEG complexeswas performed as follows: compound 1 (4.5 mg, Figure 1) wasmixed with MWCNTOX (1.7 mg) or MWCNTTEG (1.6 mg) inCHCl3 (1 mL) for 5 days and filtered through a hydrophilic PTFEmembrane. The residue was washed extensively with the washingmixture (2:2.41:1 v/vCHCl3:MeOH:water) and dried in vacuum toyield Pt(IV)@MWCNTOX and Pt(IV)@MWCNTTEG. Platinumcontent was determined using ICP-OES on the samples that wereprepared by incineration at 1000 °C followed by reconstituting theresidue in 2% HNO3.

In vivo injection of Pt@MWCNT complexes in mice

A total of 120 mice were used in the biodistribution study. Threeto four mice were employed for each formulation. Mice wereinjected via tail vein with 200 μL of CDDP aqueous solution,compound 1 in 0.5% DMSO aqueous solution, various CDDP/Pt(IV)@MWCNTcomplexes or blank nanotube aqueous suspensions.Mice treated with PBS were used as negative control. The injecteddoseswere normalized to be 4mg/kgCDDP,which is the previouslyreportedmaximum tolerated dose (MTD) of CDDP inmice,33 or 1.5mg/kg of compound 1. The injected dose of compound 1was basedon a preliminary investigation, in which mice were treated withincreasing concentrations of compound 1 to assess the MTD (datanot shown). At post-injection of 1 h, 4 h and 24 h, mice wereeuthanized for recovery of blood and removal of vital organs (brain,heart, kidney, liver, lung, spleen). Serum samples were separatedfrom blood, which was obtained by intracardiac puncture, bycentrifugation at 3000 rpm for 10 min. Tissues for elementalplatinum determination were collected and weighted for organindices (organ weight/body weight) calculation. Urine was collected

after 24 h throughmetabolic cages.Organs, serum and urine sampleswere stored at −80 °C before assays. Tissues for histopathologicalmorphology studies were fixed in 10% buffered formalin andprocessed for routine histology with hematoxylin and eosin (H&E)stain by the Institute of Molecular and Cell Biology (Singapore).Microscopic observation of tissues was carried out with an OlympusBX41 microscope (Olympus Corporation, Shinjuku, Tokyo, Japan)coupled with a digital camera.

Determination of platinum content in the organs, serum, andurine serum analysis

0.1 mL of serum samples were decomposed by the addition of0.1 mL of TMAH (25% aqueous solution) in each tube.34 Thesemixtures were shaken at room temperature for 4 days in closedcentrifuge tubes until complete digestion. The volume ofdecomposed serum was adjusted to 1 mL with deionized waterand then solutions were centrifuged at 17000 rpm for 5 min. Thesupernatant was diluted with deionized water to suitableconcentrations for analysis. Platinum concentrations of serumsamples were determined using ICP-MS.

Organs analysis

Organs (brain, heart, kidney, liver, lung, spleen) were heated at105 °C overnight for complete dehydration.34 The dried organswere decomposed by adding TMAH (25% aqueous solution) anddeionized water at a ratio of 1:1. The decomposition was carriedout at room temperature for 4 days while shaking until completedigestion. Deionized water was added to adjust the volume ofdecomposed organs to 1mL and then solutions were centrifuged at17000 rpm for 5 min. The supernatant was diluted with deionizedwater. Platinum concentrations of various organ samples weredetermined using ICP-MS.

Urine analysis

0.1 mL of urine samples were directly diluted 100-fold withdeionized water for analysis. Platinum concentrations weredetermined using ICP-MS.

ELISA assay

To determine the secretion of interleukin-1β (IL-1β), tumornecrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels inplasma, ELISA was performed using ‘Ready-SET-Go!’ kits fromeBioscience according to manufacturer’s instructions. Theabsorbance was measured on a microplate reader (TECANinfinite Series M200) at 450 nm. Results were presented as meanpg/mL ± SD of triplicates.

Statistical analysis

All statistical analyses were performed using StatisticalPackage for the Social Sciences (SPSS). Statistical comparisonswere performed using one-way analysis of variance (ANOVA).Significant ANOVAs were further analyzed using Bonferroni,Tukey post-hoc test. Differences with P values of less than 0.05were considered statistically significant.

Figure 2. TEM images of (A) pristine MWCNTs, (B) MWCNTOX, and (C) MWCNTTEG. The insets correspond to the magnification of each type of nanotube.

Table 1CDDP/Pt(IV) loading in pristine MWCNTs, MWCNTOX, MWCNTTEG and other carbon nanomaterials.

Length Diameter Pt content CDDP loading Pt(IV) loading

CDDP@MWCNT 0.3–10 μm 30–40 nm 41% 62% —CDDP@MWCNTOX 0.3–1.5 μm 30–40 nm 39% 59% —CDDP@MWCNTTEG 0.3–1.5 μm 30–40 nm 43% 58% —Pt(IV)@MWCNT 0.3–10 μm 30–40 nm 18% — 52%Pt(IV)@MWCNTOX 0.3–1.5 μm 30–40 nm 16% — 45%Pt(IV)@MWCNTTEG 0.3–1.5 μm 30–40 nm 16% — 45%CDDP@NHoxa about 40 nm 0.5–1.5 nm 12.3% 18.9% —CDDP@NHhb about 40 nm 0.5–1.5 nm 11.4% 17.6% —CDDP@SWCNTc up to several microns 1.3–1.6 nm 13.7% 21% —CDDP@US-tubesd 20–80 nm 1.4 nm 3.4–8.7% 5.2–13.4% —

a CDDP encapsulated into carbon nanohorns (NHs) with oxygen-containing functional groups at the hole edges.35,36b CDDP encapsulated into carbon nanohorns with hydrogen-terminated edges (NHh).36c CDDP encapsulated into SWCNTs.37d CDDP encapsulated into ultra-short (US) SWCNTs under different loading solvent and washing conditions.38

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Results

Characterization of MWCNTs

In this study, we present the in vivo biodistribution ofplatinum-based compounds upon their encapsulation insidevarious MWCNTs. These unfilled nanotube samples (i.e. pristineMWCNTs, MWCNTOX, MWCNTTEG) were observed by TEMprior to the entrapment of CDDP or 1 (Figure 2). The loading ofCDDP or compound 1 inside the nanotubes was determinedthrough thermal (Fig. S1) and elemental analyses. As shown inTable 1, the content of CDDP was 62% w/w in CDDP@MWCNT, 59% w/w in CDDP@MWCNTOX, and 58% w/w inCDDP@MWCNTTEG samples, respectively. These loadinglevels were slightly higher than the entrapment of 1, namely,52% w/w in Pt(IV)@MWCNT, 45% w/w in Pt(IV)@MWCNT-

OX and Pt(IV)@MWCNTTEG, likely due to the greater stericalhindrance of 1, resulting in a lower mobility within the CNTcavity.

TEM imaging and ICP-OES allowed to assess only theoverall platinum content (Fig. S2), being unable to distinguishCDDP entrapped within CNT cavity from that adsorbed on theexternal surface of the tubes. Hence, SEM was performedrepeatedly at 8–10 different positions on the grids (Fig. S3).SEM images showed no remarkable differences between pristine

MWCNTs (Fig. S3a) and CDDP/Pt(IV)@MWCNT (Fig. S3c &S3e), suggesting that there wasn’t a large amount of CDDPpresent on the surface of the CDDP/Pt(IV)@MWCNT. Similar-ly, EDX analysis on the surface of CDDP@MWCNT (Fig. S3d)and Pt(IV)@MWCNT (Fig. S3f) detected very little elementalplatinum (3.45% and 6.51% w/w, respectively), which was muchlower than the overall platinum content determined using ICP-OES, indicating that the proportion of CDDP/Pt(IV) adsorbed onthe nanotubes’ external surface was indeed very low incomparison with that entrapped within the nanotubes’ innercavity.

Tissue distribution of elemental platinum in mice

Biodistribution of elemental platinum from CDDP samplesin mice

The comparative tissue distribution study of free CDDP andCDDP entrapped within various nanotubes was investigated infemale BALB/c mice following intravenous administration ofthe maximally tolerated doses of CDDP (4 mg/kg) via tailvein.33 There was no remarkable variation of platinum content inmost tissues between CDDP alone and CDDP@MWCNTgroups. Indeed, the distribution patterns of platinum content inboth groups were similar as shown in Fig. S4. The distribution

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was further analyzed based on platinum concentrations per gramof tissues, which represent the affinity of platinum-basedmolecules for a particular tissue (Fig. S5). CDDP@MWCNTOX

and CDDP@MWCNTTEG groups exhibited higher platinumconcentrations in lungs and spleen compared with CDDP alonefollowing 4 h and/or 24 h administration (P b 0.05).

Biodistribution of elemental platinum from Pt(IV) samplesin mice

The tissue distributions of free 1 and compound 1entrapped within MWCNTs were also compared following 1h, 4 h, and 24 h post-administration (Figure 3). It presented asignificant increase of platinum content in mice exposed to Pt(IV)@MWCNTOX and Pt(IV)@MWCNTTEG groups in com-parison with 1 alone group, where the tissue distributionshowed a small amount of platinum detected in plasma,organs, and urine. On the contrary, MWCNTOX and MWCNT-

TEG not only enhanced the platinum levels in most tissuesfollowing administration of Pt(IV)@MWCNT samples, butalso altered the distribution tendency in certain tissues:platinum levels in the lungs showed the largest improvementin comparison with other tissues (Figure 3). In compound 1alone group, the platinum detected in the lungs at 1 h and 4 hpost-injection was only 0.7% and 0.2% of overall administeredplatinum, respectively. Conversely, platinum distribution in thelungs drastically increased to 14.4% at 1 h and 9.9% at 4 h inPt(IV)@MWCNTTEG group, and Pt(IV)@MWCNTOX groupalso had a remarkable increase to 4.7% at 1 h and 1.7% at 4 hpost-injection. This effect of nanotubes on distributiontendency of 1 was confirmed when platinum contents intissues were converted into the platinum concentrations pergram of tissues (Figure 4).

Figure 3. Tissue distribution of compound 1 alone, Pt(IV)@MWCNT, Pt(IV)@MWCNTOX, and Pt(IV)@MWCNTTEG in female mice at (A) 1 h post-exposure, (B) 4 h post-exposure, and (C) 24 h post-exposure. Data arepresented as mean ± SD (n = 3). *Significantly different from compound 1alone (P b 0.05).

Time-dependent variations of platinum levels in tissuesThe tissue distributions of elemental platinum at various time-

points were compared to evaluate the relationship betweenplatinum content and time in Pt(IV)@MWCNTOX and Pt(IV)@MWCNTTEG groups (Fig. S6). In Pt(IV)@MWCNTTEG group, adecreasing trend of platinum level was found in the lungs overthe time; the platinum content in the lungs dropped to 3.2% at 24h time-point, which is equivalent to about 1/5 of 1 h time-pointvalue (14.4%) and about 1/3 of 4 h time-point value (9.9%),respectively.

In contrast to the decrease of platinum levels in the lungs, Pt(IV)@MWCNTTEG group had a slight increase of platinumcontent in spleen over time, especially at 24 h time-point (Fig.S6). The variances of platinum levels in the Pt(IV)@MWCNTOX

group were not as remarkable as those in Pt(IV)@MWCNTTEG

group, even though a slight increase in platinum levels was foundin spleen and liver at 24 h time-point compared with 1 h and 4 htime-points. Unlike the striking change in the Pt(IV)@MWCNT-

TEG group, platinum level in Pt(IV)@MWCNTOX group showedonly 4.7% platinum detected in the lungs at 1 h time-point, whichwas much lower than 14.4% in Pt(IV)@MWCNTTEG group. At24 h time-point, the Pt(IV)@MWCNTOX group still maintained2.8% platinum content in the lungs, which is close to that in thePt(IV)@MWCNTTEG group.

Production of inflammatory cytokines in serum

To evaluate the effect of our samples on proinflammatorycytokine production, IL-1β, IL-6, and TNF-α levels in serum

Figure 4. Percentage of platinum dosed/gram of tissue collected from micethat were treated with compound 1 alone, Pt(IV)@MWCNT, Pt(IV)@MWCNTOX, and Pt(IV)@MWCNTTEG at (A) 1 h post-exposure, (B) 4 hpost-exposure, and (C) 24 h post-exposure. Data are presented as mean ± SD(n = 3). *Significantly different from Pt(IV) compound 1 alone (P b 0.05).

Figure 5. Serum immunological indicator levels of the control mice and theMWCNT/Pt-based drug exposed mice at 1 h, 4 h, and 24 h post-exposure.(A) IL-1β levels; (B) IL-6 levels; (C) TNF-α levels. The serum controlsamples were collected at 24 h after injection with PBS. Data are presented asmean ± SD (n = 3).

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were measured by ELISA (Figure 5). Exposure to thesecompounds and complexes induced a slight increase in IL-1βlevel in serum compared with PBS control at 1 h and 4 h post-injection (P N 0.05). The increasing trend of IL-1β level in the

CDDP/Pt(IV)@MWCNTOX and CDDP/Pt(IV)@MWCNTTEG

groups is consistent with that in the CDDP alone and compound1 alone groups, except for 4 h treatment with Pt(IV)@MWCNTOX and Pt(IV)@MWCNTTEG, which produced higher

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IL-1β level in serum. We also observed that the IL-1β level at 24h post-injection in all treated groups decreased to the same levelas PBS control (Figure 5).

In addition, no significant changes were observed in theproduction of IL-6 and TNF-α in serum (P N 0.05) for all thesamples, indicating that the injection of the drugs alone or incombination with nanotubes did not increase the IL-6 level andTNF-α level.

Histopathology of mouse tissues

Each group of treated mice was sacrificed at 24 h post-injection. The organs, including heart, lungs, liver, spleen,kidneys, and brain, were collected and processed with H&Estaining for histological observation. There were no abnormal-ities (such as necrosis, inflammation, etc.) observed in allexamined sections of tissues (Figure 6).

It was difficult to distinguish the aggregates of carbonnanotubes in tissue sections processed with H&E staining;occasionally, black aggregates were observed within Kupffercells, suggesting uptake of nanotubes (Figure S7). In order toobtain a clear background, the sections of tissues were stainedwith only eosin. The results showed that the nanotubes, whichassembled in the form of brown spots, could be found in thehistological sections from the experimental groups, while therewas no such spot in sections from the control group treated withPBS (Fig S8). It is not easy to clearly observe the nanotubes in allorgan sections because of the low amount and the inhomoge-neous dispersion of tubes in each section. However, theserepresentative staining images, correlated to the biodistributionof platinum content, suggested the presence of spread nanotubesin vital organs including heart, lungs, spleen, and kidneys,following 24 h exposure.

Discussion

Biodistribution studies were performed in order to assess theinfluence of different functionalized MWCNTs on the preferen-tial distribution of Pt-species in vivo.

Characterization of carbon nanotubes

As expected from the functionalization of our MWCNTs,MWCNTOX and MWCNTTEG were much shorter than pristineMWCNTs following 6 h oxidation, as oxidation by acidtreatment is able to cut nanotubes at the tips and at the sites ofsidewall defects (Table 1 & Figure 2). These nanotubes were stillable to retain encapsulated drug molecules inside the cavity andshowed a significant improvement in the dispersibility inaqueous environment, suggesting that they could be used as abetter carrier for drug delivery in comparison to pristineMWCNTs. Moreover, the degree of functionalization (asassessed by the Kaiser test) showed 322 μmol of NH2 groups/g of MWCNTTEG. In comparison to data in the literaturereporting that nanotubes or nanohorns were loaded with 5–21%w/w of CDDP (Table 1),35–38 higher CDDP and Pt(IV) levelswere measured inside our nanotubes (i.e. ~60% w/w of CDDP

and ~50% w/w of Pt(IV) were encapsulated into MWCNTs,respectively).

It is worth noting that the majority of the Pt detected by bothSEM and EDX analyses was in correspondence of the innerhollow space of MWCNTs, since minimum elemental platinumwas found at the tubes’ surface, thus 1) confirming that thewashing steps were successful at removing the Pt compoundunspecifically adsorbed onto the tubes’ sidewalls and 2)emphasizing high entrapment efficiencies. Therefore, ourMWCNTs could be powerful vehicles capable of carrying highdrug payload.

Tissue distribution of elemental platinum in mice

Biodistribution of elemental platinum from CDDP samplesin mice

The biodistribution study following intravenous administra-tion of the maximally tolerated doses of CDDP (4 mg/kg) andvarious nanotubes complexes (i.e. CDDP@MWCNT, CDDP@MWCNTOX and CDDP@MWCNTTEG) did not show remark-able difference (Figs. S4&S5). It is conceivable that water-soluble CDDP rapidly leaked out from the nanotubes’ innercavity when they entered the bloodstream, thus behavingsimilarly to free CDDP molecules. This result is consistentwith previous in vitro release experiments of CDDP@MWCNTin PBS medium, which indicated that most of encapsulatedCDDP was released from MWCNTs within 1 h.31

Conversely, CDDP@MWCNTOX and CDDP@MWCNTTEG

groups exhibited higher platinum content in spleen in compar-ison to CDDP alone after 4 h and 24 h administration (P b 0.05).Such disparity seemed to have a time-dependent increase: thelonger the delay from the i.v. injection, the lower theaccumulation in liver and kidney, while the higher theaccumulation in the spleen for CDDP@MWCNTOX andCDDP@MWCNTTEG samples. This tendency is probably dueto the sequestration of shorter CDDP@MWCNTOX andCDDP@MWCNTTEG by macrophages and subsequent trans-portation to spleen,39 while macrophages may have difficultiesin engulfing longer pristine nanotubes.40 Despite the fast leakageof CDDP from nanotubes at physiological conditions, a smallamount of CDDP still remained inside nanotubes and thesesamples could keep discharging CDDP after transportation to thespleen, which contributed to the slight increase of platinumconcentrations in the spleen.

Biodistribution of elemental platinum from Pt(IV) samplesin mice

A different distribution profile was found when 1 and itsentrapment within nanotubes was analyzed following 1 h, 4 h,and 24 h post-administration (Figure 3). Injection of compound 1alone group determined only a small amount of platinumdetected, while Pt(IV)@MWCNTOX and Pt(IV)@MWCNTTEG

groups showed much higher concentrations of Pt in all the tissuesinvestigated.

The low platinum level from 1 is probably attributed to itsinsolubility in aqueous solution, which might be unfavorable toits circulation in the bloodstream. This might have causedadhesion to blood vessel wall and combination with bloodproteins or cells. By contrast, MWCNTOX and MWCNTTEG

Figure 6. The histology of H&E stained liver, spleen, and kidney tissues (×20) from mice treated with CDDP alone, CDDP@MWCNTTEG, and PBS at 24 hpost-injection.

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have a more hydrophilic surface, which may enhance thedispersibility of nanotubes, thus enabling the pervasion of Pt(IV)@MWCNTOX and Pt(IV)@MWCNTTEG in physiological sys-tems as well as preventing 1 from the contact with blood vesselsand blood cells. Besides, it was expected that the stronghydrophobicity of 1, which is contrary to the water-solubleproperties of CDDP, allowed it to remain inside the CNT cavitywithout leakage in body fluid, whereas its release could bepromoted in the presence of reducing agents inside the cells via adrastic reversal in hydrophobicity.32 Therefore, most of 1 wasable to enter systemic circulation along with MWCNTOX andMWCNTTEG rather than behaving like free molecule, suggestingthat MWCNTOX and MWCNTTEG had a greater impact on thetissue distribution of 1 than of CDDP.

Moreover, Pt(IV)@MWCNTOX and Pt(IV)@MWCNTTEG

complexes also affected the preferential distribution of Pt intospecific organs: in Pt(IV)@MWCNTOX and Pt(IV)@MWCNT-

TEG groups, platinum concentration in the lungs was much higherthan in the other tissues at 1 h and 4 h post-injection, whereas thehighest platinum concentrations were detected in the kidney andliver when mice were treated with free 1. This variance suggeststhat delivery by MWCNTOX and MWCNTTEG seems favorableto the transportation of platinum-based drugs to the lungs forlung carcinoma treatment. This result is in good agreement withprevious distribution studies of oxidized MWCNTs in mice,showing that most oxidized MWCNTs accumulated in the lungs,with just a small amount of retention in the liver and spleen afterintravenous administration.41 It was considered that pulmonarycapillary bed captured oxidized MWCNTs and subsequently

retained them in the lungs, but a little part of oxidized MWCNTscould be cleared and could enter into the circulatory system.Also, the oxidative treatment used to prepare MWCNTOX andMWCNTTEG reduced the length of the nanotubes and theseshorter nanotubes could be readily taken up by alveolar epithelialcells. In contrast, the hydrophobic pristine nanotubes wouldassemble as long bundles in physiological environment; theselarge aggregates of nanotubes would prevent them from beingengulfed by cells and decrease the affinity for the lungs.42–44

Platinum-based antitumor drugs are currently used as first-linechemotherapy for non-small-cell lung cancer (NSCLC),45,46

whereas clinical studies suggested that reduced platinumaccumulation might be an important mechanism of platinumresistance, which is a major limitation in the treatment ofadvanced NSCLC.47 Therefore, functionalized-MWCNTs couldbe a promising carrier to achieve higher accumulation oftherapeutic molecules in the lungs with reduced adverse effectson normal tissues.

In proportion, platinum content in the kidney decreased themost among all the tissues when compound 1 was entrappedwithin MWCNTOX and MWCNTTEG. Indeed, nephrotoxicity isone of the most common side effects associated with platinum-based chemotherapeutics.13,48 Figure 4 shows that the kidneyhad the highest platinum concentration in compound 1 alonegroup, while platinum levels in the lungs, liver and spleentranscended that in kidneys when mice were treated withfunctionalized-MWCNTs. This variation suggests that deliveryby MWCNTOX and MWCNTTEG might be helpful in reducingthe nephrotoxicity of platinum-based drugs.

9J. Li et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2014) xxx–xxx

Time-dependent variations of platinum levels in tissuesAlthough Pt(IV)@MWCNTTEG group showed much higher

platinum content in the lungs compared with pristine Pt(IV)@MWCNT and Pt(IV)@MWCNTOX groups, MWCNTTEG inter-nalized in the lungs probably promoted their clearance bymacrophages, thus resulting in the fast decrease of platinum levelin the lungs. Once again, this result suggests that MWCNTTEG

might be useful at promoting drug release to the lungs whileavoiding an excessive accumulation in the target organ. Futurestudies could address this clinically relevant aspect, i.e. theoptimal transition time of platinum-based drugs in the lungs for adesirable therapeutic effect against NSCLC.

It was also noted that Pt(IV)@MWCNT complexes wereresponsible for an increase of platinum in spleen over time,especially after 24 h (Figure 3). This increase is in accord withthe observations reported in the biodistribution studies ofCDDP encapsulated into liposomes,49,50 where the maximumCDDP uptake in spleen was achieved at 1 h and 12 h post-injection with free CDDP and stealth pH-sensitive liposomescontaining cisplatin, respectively. Similarly, intravenouslyadministrated nanotubes, as exogenous materials, might activatethe immune response. Hereby, these nanotubes distributedthroughout the body were likely trapped in alveolar macro-phages and Kupffer cells, and then transported to lymph nodesand spleen.20 Al-Jamal et al. observed translocation offunctionalized MWCNTs from lung to spleen,30 which isexplained by the transportation of overloaded MWCNTs fromthe lungs to the afferent lymphatics and then to the spleen.27,28

On the other hand, a great number of nanotubes would bedirectly captured by RES (e.g. sinus histiocytes in spleen),resulting in the enhanced uptake of nanotubes into the spleen.41

Spleen retention of nanotubes was expected to be subsequentlycleared through the renal pathway.51 This behavior iscomparable to the findings reported by Wu et al. showingthat the dose of Fe in the spleen did not reach a maximum until8 h after intravenous injection of MWCNT/Fe3O4 hybrid, andsubsequently its concentration in the spleen decreasedsignificantly.52 Therefore, the increase of platinum level inthe spleen at the primary stage of clearance is expected in viewof the spleen’s major role in immunomodulation. A similarincrease of platinum level at 24 h post-injection was observed inthe liver, which is also a RES organ, in both Pt(IV)@MWCNTTEG groups and Pt(IV)@MWCNTOX.

On the other hand, the accumulation of platinum contentfrom Pt(IV)@MWCNTOX group in the lungs at 1 h time-point (Figure 3, A) was much lower (4.7%) than 14.4% in Pt(IV)@MWCNTTEG group. Presumably, such low level ofnanotubes accumulation/deposition in the lungs likely avoidedthe intense immune response and escaped from rapidphagocytosis by macrophages, while acute eliminationresponse might be activated upon the accumulation ofnanotubes to a higher extent.53 However, the amount detectedat 24 h time-point was similar between the 2 groups (Figure 3,C): we postulated that the clearance rate of Pt(IV)@MWCNTOX and Pt(IV)@MWCNTTEG from the lungs mightbe correlated with the amount internalized in this specificorgan, while their final retention was comparable over thesame period of time (i.e. 24 h).

Production of inflammatory cytokines in serum

The evaluation of potential pro-inflammatory effects causedby the administration of our Pt-compounds (either as isolatedmolecules or as complexes with functionalized MWCNTs)showed an increase in IL-1β level across all the samples, exceptfor 4 h treatment with Pt(IV)@MWCNTOX and Pt(IV)@MWCNTTEG, which produced higher IL-1β level in serum.This difference is probably associated with the persistent releaseof platinum-based compound from functionalized-MWCNTs, aswell as the resulting higher platinum concentrations in overalltissues compared with the compound 1 alone group. Anyway,the IL-1β level at 24 h post-injection in all treated groups showeda decrease and became comparable to the PBS control. Thisdecrease might be attributed to the clearance of platinum-basedcompounds from mice, which has been shown in thebiodistribution study (Figure 5). We thereby postulated that thestimulation for IL-1β production mainly stemmed from theinjection of platinum-based compounds. Moreover, MWCNTOX

and MWCNTTEG as carriers did not result in any increased effecton the IL-1β production (data not shown), suggesting that thefunctionalized-MWCNTs themselves did not induce any addi-tional inflammatory reaction once injected in mice. Moreover,the fact that no remarkable changes were observed in theproduction of IL-6 or TNF-α in serum (P N 0.05) for all samplesis coincident with the results that no treatment-related micro-scopic findings were noticed in additional histopathologicalinvestigations (vide infra).

Histopathology of mouse tissues

Histological examination of the sectioned tissues (heart,lungs, liver, spleen, kidneys, and brain) did not show anyapparent abnormality such as necrosis or inflammation (Figure6). We postulated that such good biocompatibility of nanotubesand CDDP/Pt(IV)@MWCNT complexes in our study was likelydue to the low-dose and short-period exposure, since the tissueinjuries caused by nanotubes were both dose- and time-dependent.54 Therefore, neither nanotubes as carriers nor theCDDP/Pt(IV)@MWCNT complexes induced acute toxicity tomice during 24 h exposure.

Conclusion

In this study, CDDP and compound 1 were entrapped withinpristine MWCNTs, MWCNTOX, and MWCNTTEG to obtain ahigh drug loading inside the cavity of nanotubes. In order toinvestigate the effects of various nanotubes on the biodistributionof platinum-based compounds, female BALB/c mice wereexposed to free CDDP or 1, as well as CDDP/Pt(IV)@MWCNT complexes. The results showed that functionalizedMWCNTs could significantly enhance the platinum content inalmost all tissues and efficiently alter the tissue distribution inmice for the delivery of Pt(IV) compound. In addition, thenanotubes as drug carriers did not induce any additionalinflammatory reaction or necrosis when injected in mice.Functionalized nanotubes could be exploited as a promising

10 J. Li et al / Nanomedicine: Nanotechnology, Biology, and Medicine xx (2014) xxx–xxx

nano-carrier to improve accumulation of drug molecules in thelungs for therapeutic treatments.

Appendix A. Supplementary data

Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.nano.2014.01.004.

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Graphical Abstract

Nanomedicine: Nanotechnology, Biology, and Medicine xxx (2014) xxxIn vivo biodistribution of platinum-based drugs encapsulatedinto multi-walled carbon nanotubes

Jian Li, PhD a, Aakansha Pant a, Chee Fei Chin b, Wee Han Ang, PhD b,*,Cécilia Ménard-Moyon, PhD c, Tapas R. Nayak, PhD a, Dan Gibson, PhD d,Sundara Ramaprabhu, PhD e, Tomasz Panczyk, PhD f, Alberto Bianco, PhD c,*,Giorgia Pastorin, PhD a,g,h,*

aDepartment of Pharmacy, National University of Singapore, Science Drive 2, SingaporebDepartment of Chemistry, National University of Singapore, SingaporecCNRS, Institut de Biologie Moléculaire et Cellulaire, Laboratoire d’Immunopathologie etChimie Thérapeutique, Strasbourg, FrancedSchool of Pharmacy, The Hebrew University of Jerusalem, IsraeleDepartment of Physics, Indian Institute of Technology Madras, Chennai 600 036, IndiafInstitute of Catalysis and Surface Chemistry, Polish Academy of Sciences ul.Niezapominajek 8, 30239 Cracow, PolandgNUS Graduate School for Integrative Sciences and Engineering, Centre for Life Sciences(CeLS), 28 Medical Drive, SingaporehNUSNNI-NanoCore, National University of Singapore, T-Lab Level 11, 5A EngineeringDrive 1, Singapore

The chemotherapeutic drug cisplatin or its derivative platinum(IV) complex wereentrapped inside functionalized-multi-walled carbon nanotubes and then intravenouslyinjected into mice to investigate the influence of the nanotubes on the biodistributionof Pt-based molecules. The amino-functionalized nanotubes significantly enhancedthe accumulation of Pt(IV) sample in the lungs, and reduced both kidney andliver accumulation.

Nanomedicine: Nanotechnology, Biology, and Medicinexx (2014) xxx

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