Hyaluronate Tethered, “Smart” Multiwalled Carbon Nanotubesfor Tumor-Targeted Delivery of DoxorubicinSatyajit R. Datir, Manasmita Das, Raman Preet Singh, and Sanyog Jain*

Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education andResearch (NIPER), Sector 67, SAS Nagar (Mohali) Punjab, India 160062

*S Supporting Information

ABSTRACT: The present study reports the optimized synthesis, physicochemical characterization, and biological evaluation of anovel, multiwalled carbon nanotube-hyaluronic acid (MWCNT-HA) conjugate, complexed with an anticancer agent,Doxorubicin (DOX) via π−π stacking interaction. The therapeutic conjugate was concomitantly labeled with a near-infraredfluorescent dye, Alexa-Flour-647 (AF-647), and radiotracer Technetium-99m (99mTc) to track its whereabouts both in vitro andin vivo via optical and scintigraphic imaging techniques. Covalent functionalization of MWCNTs with HA facilitated theirinternalization into human lung adenocarcinoma, A549 cells via hyaluronan receptors (HR) mediated endocytosis. Internalizednanotubes showed lysosomal trafficking, followed by low pH-triggered DOX release under endolysosomal conditions.Consequently, DOX-loaded HA-MWCNTs exhibited 3.2 times higher cytotoxicity and increased apoptotic activity than freeDOX in equivalent concentrations. Organ distribution studies in Ehlrich ascites tumor (EAT) bearing mice model indicated thattumor specific localization of 99mTc-MWCNT-HA-DOX is significantly higher than both free drug and nontargeted MWCNTs.Pharmacodynamic studies in chemically breast-cancer-induced rats showed that the tumor-growth inhibitory effect of HA-MWCNT-DOX was 5 times higher than free DOX in equivalent concentration. DOX delivered through HA-MWCNTs wasdevoid of any detectable cardiotoxity, hepatotoxicity, or nephrotoxicity. All these promising attributes make HA-MWCNTs a“smart” platform for tumor-targeted delivery of anticancer agents.

Carbon nanotubes (CNTs) represent a novel class of nano-materials, which made their appearance in the nano-

medicine arena, not more than a decade ago. Ever since theiremergence on the nanoplatform, their unique physicochemicalproperties such as high aspect ratio and surface area, highmechanical strength, ease of drug loading via π−π stackinginteractions, and photoacoustic effect have made them potentialcandidates for targeted drug delivery and imaging. A majortechnological barrier, which obscures the application of pristineCNTs in biomedicine, is their lack of solubility in aqueousmedia. Hydrophobicity of CNTs, coupled with van der Waalsand strong π−π interactions between the individual tubes,cause pristine CNTs to assemble as bundles, rendering themnonbiocompatible and highly toxic. Functionalization of CNTs,therefore, is crucial for their assimilation into multifarioussystems for industrial as well as biomedical applications. One ofthe most powerful approaches for rendering CNTs soluble inaqueous medium is the covalent functionalization of their side

walls and tips. Functionalized CNTs ( f-CNTs)1−4 can easilycross the cell membrane facilitating the internalization of vari-ous cargos inside the cells (e.g., fluorescein, plasmid DNA,proteins, etc.) that would not otherwise be taken up. In vivobiodistribution studies further confirm that well functionalizednanotubes (f-CNTs) circulate in blood with half-lives on theorder of a few hours.1,5 Recent expansion in functionalizationmethods has also stimulated the growth of multifunctional,drug-loaded CNTs combined with the identification of disease-specific molecular target and imaging capabilities.4,6,7 Covalentfunctionalization of CNTs with tumor targeting componentssuch as antibodies, peptides, or small molecules (viz. folic acid)promises to expand the therapeutic window for a variety of

Received: May 8, 2012Revised: October 3, 2012

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anticancer drugs.7,8 Although targeted delivery of anticanceragents using active-targeted CNTs has been investigated byvarious research groups, most of these studies are limited to thein vitro evaluation of their targeting or therapeutic efficacies.For example, Heister et al. have demonstrated that oxidizedsingle-walled carbon nanotubes (SWCNTs) trifunctionalizedwith the anticancer drug Doxorubicin, a monoclonal antibody,and a fluorescent marker are efficiently taken up by cancer cellswith subsequent translocation of the drug to the nucleus whilethe nanotubes remain in the cytoplasm.8 Similarly, Dhar et al.have used folate as a homing device for SWCNT mediated Pt(IV) prodrug delivery.7 Nevertheless, none of these reportshave included any study to evaluate the antitumor efficacies ofthese functionalized CNTs in vivo. From the synthesis pointof view, development of multifunctional CNTs, tethered withmultiple diagnostic and therapeutic entities, is highlychallenging, and even more challenging is their precise char-acterization and biological evaluation. The present work thusstemmed from the need to contribute some new toolboxes forthe fabrication of multifunctional CNTs and subsequentlyevaluate their theragnostic potential in vivo.Hyaluronic acid (HA) is a well-known, naturally occurring

glycosoaminoglycan, composed of repeating disaccharide unitsof D-glucuronic acid and (1-β-3)-N-acetyl-D-glucosamine.Activated hyaluronate receptors (HR) are ubiquitously overex-pressed by CD44 and receptor for hyaluronan-mediated moti-lity (RHAMM) on tumor cells9 that are lacking in theirnon-tumorigenic counterparts. The unique ability of CD44 to inter-nalize HA reinforces its potential as a targeted drug deliveryvehicle. HA generates a key signal for activating the kinasepathways and regulating angiogenesis in tumors. This uniqueproperty of HA has been previously recognized, and severalpreclinical studies have chemically conjugated HA with a broadspectrum of cytotoxic agents, concluding with highly effectiveanticancer agents in vitro.10 The present study reports theoptimized synthesis of a novel multiwalled carbon nanotube−hyaluronic acid conjugate, complexed with the anticancer drug,Doxorubicin, via π−π stacking interaction. In addition to pre-cise characterization of the conjugate, extensive in vitro and invivo studies were undertaken to explore how conjugation of HAwith MWCNTs influences the tumor-targetability and ther-apeutic efficacy of CNT-entrapped DOX against HR positivecancer cells. A near-infrared (NIR) fluorescent dye Alexa-Flour-647 (AF-647) and radiotracer Technetium-99m (99mTc) werefurther integrated with DOX-loaded HA-MWCNTs to enablereal-time tracking of the localization of CNT−drug conjugatesthrough optical and/or scintigraphic techniques. To the best ofour knowledge, this is the first report in which HA-targetedMWCNTs have been successfully exploited for tumor-selectiveintravenous delivery of DOX, while reducing the drug-associatedcardiotoxicity.

■ MATERIAL AND METHODSMaterials. Multiwalled carbon nanotubes (purity >95%,

length 1−2 μm, and diameter 20−30 nm) were procured fromNanovatech Pvt. Ltd., USA. Sulfuric acid, nitric acid (69−72%),disodium hydrogen phosphate, sodium acetate, thionyl chlo-ride, sodium lauryl sulfate, copper sulfate, and thiobarbituricacid were purchased from Loba Chemie Pvt. Ltd., Mumbai,India. Doxorubicin and hyaluronic acid were obtained as giftsample from Sun Pharmaceuticals, India, and FocaschemBiotech limited, China, respectively. 2,2′-(Ethylene dioxy) bis-(ethylene amine), PEG-bis-amine 3000, propidium iodide (PI),

neutral red (NR), Alexa-Fluor 647, and 7,12 dimethylbenz-[α]anthracene (≥95% pure) were purchased from Sigma, USA.Annexin V staining kit, 7,12-dimethylbenz[α]anthracene(DMBA; ≥95% pure), 2,2′-(ethylene dioxy) bis(ethyleneamine), poly(ethylene glycol) PEG-bis-amine Mw 3000, PEGbis(amine) Mw 6000, propidium iodide (PI), and Rhodamine123 (Rh123) were purchased from Sigma, USA. All kits forbiochemical estimations were procured from Accurex, Bio-medical Ltd., Mumbai. Culture medium and serum were pro-cured from PAA, Austria. Lung epithelial cancer cell line A549was purchased from National Centre for Cell Sciences, Pune,India. AF-647 was purchased from Invitrogen, USA. TUNELassay kit was purchased from Calbiochem, USA. All otherchemicals and solvents were of analytical grade and procuredfrom local suppliers unless otherwise mentioned.

Functionalization of MWCNTs. Functionalization ofCNTs with hyaluronic acid (HA) and drug loading was accom-plished in the following steps: (i) Surface oxidation of pris-tine MWCNTs; (ii) acylation of acid-oxidized carboxylatedMWCNTs; (iii) amine functionalization of acylated MWCNTsvia appropriate PEG spacer; (iv) HA conjugation with amine-functionalized MWCNTs; and (v) DOX loading via π−π stack-ing interaction.

Surface Oxidation of Pristine MWCNTs. For surface oxida-tion, pristine MWCNTs (200 mg) with approximate lengthranging 1−2 μm and diameter 20−30 nm were dispersed in a3:1 (v/v) mixture of H2SO4/HNO3 (20 mL) via ultrasonicationfor 5 min. Thereafter, the dispersion was refluxed at 80 °Cusing a hotplate cum magnetic stirrer at 900 rpm. Oxidationwas continued for 3 h. After oxidation, the suspension ofMWCNTs in acid was diluted up to 5 times of its volume andcentrifuged at 21 000 rpm. This washing was repeated at leasttwice for complete removal of acid. Oxidized MWCNTs ob-tained were further dispersed in acetone and centrifuged,following which the supernatant was discarded and MWCNTswere air-dried for approximately 12 h to obtain fine powders ofoxidized CNTs.11 The transformation of pristine to oxidizedCNTs was almost quantitative (yield ∼95% w/w).

Amine Functionalization of Oxidized MWCNTs. For aminefunctionalization, oxidized MWCNTs (100 mg) were dispersedin THF (10 mL) via ultrasonication for 1 min. To the resultantdispersion, SOCl2 (15−20 mL) was added, and the mixture wasrefluxed at 80 °C for 24 h.12 Thereafter, solvents were removedusing rotavapor and the resultant acylated MWCNTs weredispersed in anhydrous THF. From thermogravimetric analysis(TGA), carboxylic density on the surface was determined to be0.0018 mmol/mg of CNTs. This value was necessary to cal-culate the exact amount of PEG-3000 or EDBE that would beideally required for complete interchange of surface carboxylgroups with 2,2′-(ethylene dioxy) bis(ethylene amine)(EDBE). On the basis of TGA results, around 3-fold molarexcess of PEG-bis-amine (16.2 mg/mg of oxidized MWCNTs)or EDBE (0.810 μg/mg of oxidized MWCNTs), dissolved inanhydrous DMSO was added to a suspension of acylatedMWCNTs in a 5:1 (v/v) mixture of DMSO and pyridine underice-cold conditions. The reaction mixture was left stirring for12 h, following which the reaction mixture was subjected tocentrifugation, supernatant discarded, and the pellet of EDBE-MWCNTs/PEG 3000-MWCNTs was washed with distilledwater, recentrifuged to remove the unreacted reagents, anddried by repeated washing with acetone. Surface amine densityof functionalized MWCNTs was determined using the p-nitro-benzaldehyde, colorimetric assay (see Supporting Information,

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Methods Section, for further details). The transformation ofoxidized CNTs to its amine functionalized counterpart wasnearly quantitative (yield ∼95% w/w).Conjugation of Hyaluronic Acid (HA) with Amine-Function-

alized MWCNTs. For conjugation of HA with MWCNTs, HA(taken in a concentration equivalent to the surface aminedensity of MWCNTs) was dissolved in water at pH 8.0 andactivated with N-hydroxy succinimide (NHS) and EDC.HClfor 6 h. To this activated HA, an aqueous suspension of EDBE-MWCNTs was added and the reaction mixture was stirred for24 h. Subsequently, HA-EDBE-MWCNTs were separated andair-dried for 12 h (yield ∼80% w/w).Physicochemical Characterization of Synthesized

Conjugate Bound to MWCNTs. Particle Characteristicsand Surface Chemistry. Microstructure of f-MWCNTs wasanalyzed using scanning electron microscopy (SEM, modelS30400) and transmission electron microscopy (TEM, modelFEI Tecnai G2), while hydrodynamic sizes and zeta potential ofthe nanotubes in water were measured using the Malvern ZetaSizer (Nano ZS, Malvern Instrument, USA). Surface chemistryof the functionalized nanotubes was studied using Fouriertransform infrared (FTIR) and thermogravimetric analysis(TGA), while fine structure resolved analysis of the surface-bound ligands was performed using high-resolution magic anglespinning NMR (HRMAS-NMR) spectroscopy. FTIR spectrawere recorded on Perkin-Elmer systems using KBr pellets andprocessed using Spectrum software. TGA was carried out onMettler Toledo system (TGA/SDTA 851e) by heating CNTsat the rate of 10 °C/min. Samples for HRMAS NMR experi-ments were prepared by suspending 10 mg of each nanoparticlepreparation in a 1:1 mixture of DMSO-d6/D2O (500 μL).HRMAS−NMR analysis was carried out with a 400 MHz FT-NMR spectrometer (Avance 400), equipped with a 5 mmHRMAS probe.DOX Loading on MWCNTs. For DOX loading on

MWCNTs, HA-MWCNT/DOX were mixed in the ratio of2:1 (w/w), and the resultant mixture was incubated at fourdifferent pH values: 5.5, 7.4, 8.0, and 9.0. The CNT−drugmixture was kept in a shaker bath at 37 °C for 24 h, followingwhich drug-loaded CNTs were separated by centrifugation andabsorbance of the supernatant was measured at 480 nm. Drugloading was calculated spectrophotometrically using thefollowing formula: % drug encapsulation efficiency = (A1 −A2)/A1 × 100, where A1 = free Doxorubicin absorbance andA2 = absorbance of supernatant after centrifugation. Percentage(%) dug loading was estimated as % drug loading = amount ofdrug encapsulated/weight of the pellet × 100.pH-Dependent Release of DOX from MWCNTs. The release

behavior of DOX from HA-MWCNTs was checked at twodifferent pH conditions, viz., pH 7.4 phosphate buffered saline(15 mmol) and pH 5.5 acetate buffered saline (15 mmol),to mimic the physiological and lysosomal pH, respectively.Briefly, an aqueous dispersion of drug-loaded MWCNTs (5 mgdispersed in 2 mL water) was used to fill the dialysis membrane(MWCO 12 000 Da), poured into release medium (15 mL),and placed inside the water shaker bath for 48 h at 37 °C and1200 rpm. Aliquots were taken at periodic intervals up to 48 h,and absorbance was recorded using UV spectroscopy at480 nm.13 The stability of HA-MWCNT-DOX in serum wasdetermined by incubating the nanotubes in 100% fetal bovineserum (FBS) at 37 °C. Released DOX was removed by filtra-tion at different time points, and absorbance was recordedusing UV spectroscopy at 480 nm.

In Vitro Cellular Uptake, Cytotoxicity, and ApoptosisStudies. Cell Culture. Cell uptake studies were conducted inA549 lung cancer cell lines obtained from the National Centrefor Cell Sciences (NCSS) Pune. All cell lines were grown inDulbecco’s modified Eagle’s medium supplemented with 10%(v/v) heat inactivated fetal calf serum 2 mM glutamine, 100units mL−1 penicillin 100 μg/mL streptomycin, 4 mmol L−1

glutamine at 37 °C in a 5% CO2 and 95% air humidified atmo-sphere. Confluent cultures were harvested by trypsinization andcells counted and suitably diluted to obtain 5 × 105 cells/mL.Cell suspension (200 μL) was added in 96 well tissue cultureplates and incubated overnight for cell attachment. Cellsuspension (5 × 105 cells/mL; 200 μL) was added in 96 welltissue culture plates and incubated overnight for cell attach-ment. For all cell culture experiments, CNTs were dispersed inculture medium to obtain a stock dispersion of 1 mg/mL. Thestock dispersion was bath sonicated for 5 min to aid indispersion. This stock dispersion was serially diluted in culturemedium to achieve the desired CNT concentrations.

In Vitro Cell Uptake and Colocalization Studies. For com-petitive inhibition studies, A549 cells were exposed to 100 μg/mL of different nanotube preparations in the absence andpresence of HA (50 μg/mL).14 After 3 h of incubation, the cellswere thoroughly washed to remove the non-internalized nano-tubes and confocal laser scanning microscopy (CLSM) wasperformed using model Olympus FV 1000. Intracellular colo-calization of CNTs was examined by labeling lysosomes andmitochondria of A549 cells with neutral red (NR) and Rhodamine123 (Rh 123), respectively, as described in our previous reports.15

Subcellular distribution of the nanotubes was visualized throughconfocal microscopy.

In Vitro Cytotoxicity and Apoptosis Study. f-MWCNT-induced cytotoxicity in A549 cells was determined by MTTassay using our previously published protocol.16The inductionof apoptosis was determined by phosphatidylserine external-ization (Annexin V staining) and DNA fragmentation (TUNELassay) after 24 h incubation using commercial kits according tomanufacturer’s instructions. Nuclear morphology was assessedafter 24 h incubation with free DOX and CNTs. Cells werefixed in 70% ethanol and stained with 10 μg/mL PI. DNAfragmentation was determined by diphenylamine (DPA)method as described elsewhere.17

In Vivo Studies. Biodistribution Studies. Biodistribution ofthe 99mTc-free DOX and MWCNT−DOX was studied in anEhlrich ascites tumor (EAT) bearing mice model. The protocolfor biodistribution studies was duly approved by the Institu-tional Animal Ethics Committee (IAEC) of Institute of NuclearMedicine and Allied Science (INMAS), Delhi. The tumor wasimplanted into the mice by injecting 0.1 mL of cell suspensionof EAT cells subcutaneously in the right hind paw of the mice.The injected volume contained approximately (1.0−1.5) × 107

cells. Tumor was allowed to grow for 10 days. The mice weredivided into 3 groups of 16 animals each (total 48 mice, 4 miceeach for one time interval). Each mouse received 100 μCi(100 μL) doses of the labeled formulations by tail veininjection. The mice were humanely sacrificed at 1, 4, and 24 hafter the injection. The blood was collected by cardiac punc-ture. Subsequently, major organs/tissues were dissected/harvested, washed with Ringer’s solution to remove any adherentdebris, and dried using tissue paper. The organs were taken inpreweighed tubes, which were then weighed again to calculatethe weight of organ/tissue, and radioactivity corresponding tothem was measured using a well-type γ-scintillation counter.

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The results were expressed as the percentage of injected dose(ID) per gram of organ.Tumor Growth Inhibition Studies. Female Sprague−Dawley

(SD) rats of 220−230 g and 4−5 weeks old were suppliedby the central animal facility (CAF), NIPER, India. Forpharmacodynamics study, all animal study protocols wereduly approved by the Institutional Animal Ethics Committee(IAEC) of the National Institute of Pharmaceutical Education& Research (NIPER), India. The animals were acclimatized attemperature of 25 ± 2 °C and relative humidity of 50−60%under natural light/dark cycle for one week before experiments.For tumor induction, 7,12-dimethylbenz[α]anthracene(DMBA) in soybean oil was administered orally to rats at45 mg/kg dose at weekly interval for three consecutive weeks.Measurable mammary tumors were observed in animals after10 weeks of last DMBA dosing, and tumor-bearing animalswere separated. For pharmacodynamic assessment, animalswere divided into five groups, each group containing fouranimals. Free DOX, DOX-deprived MWCNT-EDBE-HA,MWCNT-EDBE-DOX, and MWCNT-EDBE-HA-DOX wereadministered to the first four groups through a single dose of5 mg/kg of plain DOX and MWCNTs in equivalent concen-tration of the free drug via intravenous injection. The last groupwas kept as a control, which received normal saline in a similarway. The tumor volume and body weight was measured onevery alternate day post-treatment by Vernier caliper using thefollowing equation:

= ×V D dtumor volume ( ) /22

where d is the smallest and D the longest length of the tumor.The study was terminated after 10 days post-treatments.

Blood was collected through cardiac puncture for analyzingbiochemical markers of toxicity.Toxicity Studies. In order to address the toxicity issues

pertaining to free DOX and CNTs, toxicity studies were carriedout in tumor-induced rats as well as in normal mice, which weretreated with free DOX/functionalized MWCNTs at the dosesimilar to that used in efficacy studies. Toxicity studies wereconducted in both rats and mice because compared to rats,mice present more sensitive models for toxicity evaluation.Cardiotoxicity of DOX-loaded f-MWCNTs was determinedin breast tumor-induced Sprague−Dawley (SD) female rats.Prior to sacrifice of animals treated with free DOX andvarious MWCNT preparations for 10 days, blood sampleswere collected via cardiac puncture into heparinized capillarytubes. Plasma was separated by centrifugation at 10 000 RCFfor 10 min and stored at −20 °C until analysis. Enzymeactivities such as CK-MB (creatine phosphokinase) andLDH (lactate dehydrogenase) levels were analyzed inplasma, while malondialdehyde (MDA) and superoxidedismutase (SOD) were determined in heart homogenateby the commercially available kits based on the methodprovided by the manufacturer instructions supplied withthe commercial kits. For cardiotoxicity evaluation in mice,animals were divided into 4 groups with each groupcontaining 6 animals. Cardiotoxicity in mice was evaluatedfollowing the same protocol as described in the case oftumor-bearing rats. Hepatotoxicity and nephrotoxicity inmice was evaluated following the protocol reported in ourearlier publication.18 The detailed procedure for thedetermination of various biochemical markers, viz., AST,ALT, MDA, SOD, have also been detailed in the SupportingInformation.

Statistical Analysis. All data unless otherwise specified areexpressed as mean ± SD. Statistical analysis was performedwith Graph Pad Prism software (v 4.03, USA) using one-wayANOVA, followed by Tukey−Kramer multiple comparisontest. P < 0.05 was considered statistically significant.

■ RESULTS AND DISCUSSIONSynthesis, Optimization, and Characterization of HA-

Functionalized MWCNTs. Functionalization of MWCNTswith Hyaluronic Acid. A schematic representation of thefunctionalization of MWCNTs with hyaluronic acid has beendepicted in Figure 1. The conjugation of different functionalmolecules on MWCNTs was qualitatively analyzed using FTIRand HRMAS-NMR analysis (Figure S1, S2). The quantity ofvarious functional molecules immobilized per unit mass ofCNTs was determined using TG analysis (Figure S3, TableS1). As a preliminary step toward the multiple functionalizationof MWCNTs, pristine MWCNTs having approximate lengthbetween 1 and 2 μm and diameter 20−30 nm were enrichedwith surface carboxyl groups via oxidation in the presence ofconcentrated H2SO4 and HNO3 (3:1 v/v) for 3 h. The carboxylfunctions on MWCNTs were interchanged with amine groupsthrough a sequence of thionyl chloride (SOCl2) inducedacylation (24 h) and further reaction with around 3−5-foldmolar excess of an appropriate PEG-bis amine. In order toensure maximum interchange of surface carboxyl groups withsurface-pendant amine groups, the PEGylation process wasinitially optimized in terms of spacer length and molecularweight. Acylated MWCNTs were reacted with a variety ofPEG-bis amine ranging from the mini-PEG 2,2′-(ethyl-enedioxy) bis(ethylamine) [EDBE, Mw = 148.2] to PEG-bis-amine withMw 3000 or 6000. In the case of EDBE, the majorityof the surface carboxyl groups could be interchanged withamine groups as evident from the high surface amine density(0.458 μmol/mg) of EDBE-MWCNTs determined viap-nitrobenzaldehyde assay. On the contrary, surface aminedensity of MWCNTs functionalized with PEG 3000 or 6000was even less than 0.1 μmol/mg of CNTs. Zeta potentialanalysis further revealed that transformation of acylatedMWCNTs to EDBE-MWCNTs was accompanied withcomplete reversal of surface polarity from −57.5 ± 2.7 mV to+9 ± 0.6 mV. The changes in surface charge as well as aminedensity were perceived within one hour of addition of thereagents, whereas in the case of PEG-bis-amine 3000, even after24 h of reaction, zeta potential of the CNT surface wascompletely negative (−28.6 ± 6.6 mV). The observation maybe attributed to any one of the following factors: First, due tothe large size of the polymer, it is quite feasible that only afraction of the total number of COO− groups on the surfacewere PEGylated. Further, it may also happen that the longPEG-Bisamine chain folds and the free amino terminalassociated with the polymer binds with the unreacted COO−

groups on oxidized MWCNTs via electrostatic interaction. Inaddition, amine functionalization with EDBE is more economicand cost-effective as compared to its higher MW counterparts.Stoichiometric calculations revealed that 1 mg of carboxylatedMWCNTs requires at least 5.4 mg of PEG-bis-amine 3000,which is approximately 20 times higher than the amount ofEDBE (270 μg) required for complete interchange of allcarboxyl groups on acid-oxidized MWCNTs with aminegroups. Considering the distinct advantages of EDBE ascompared to its higher Mw counterparts, EDBE-MWCNTswere chosen as the preferable intermediate for further

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bioconjugation reactions. To conjugate HA with EDBE-MWCNTs, HA was activated with 1.5-fold molar excess of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) inaqueous solution (pH ∼8) and subsequently reacted withEDBE-MWCNTs for 48 h. The bioconjugation step wasaccomplished with two types of HA: high Mw HA (∼120 kDA)and low Mw HA (∼5 kDA). As surface amine density ofMWCNTs was determined to be around 0.458 μmol/mg,160 μg HA/mg CNTs and 1.39 mg HA/mg CNTs were,respectively, required for the complete derivatization of thesurface amine groups of CNTs with HA. Considering therelative cost of high and lowMw HA, the use of lowMw HA wasfound to be more effective. Moreover, high Mw HA tends toform meshwork around the CNTs and increases their effectivehydrodynamic size, which negatively influence the nanotube−drug interaction and takes away from their effectiveness forintravenous delivery.Particle characteristics of acid-oxidized carboxylated MWCNTs,

EDBE-MWCNTs, and HA-EDBE-MWCNTs have been sum-marized in Table 1. Oxidized MWCNTs present an averagehydrodynamic size and zeta potential of 173.8 ± 4.2 nm and−57.5 ± 2.7 mV, respectively. Following functionalizationwith EDBE, the average hydrodynamic size of CNTs increasedwith concomitant reversal of zeta potential, i.e., the EDBE-MWCNTs became positively charged due to the presence offree amine groups on their surface. After HA conjugation, thehydrodynamic size of the nanotubes increased to 451.3 ± 8.7 nm

and zeta potential again shifted to the negative direction(−42 ± 4 mV) due to the covalent attachment of polyanionicspecies on the surface of CNTs. Representative SEM image ofvarious MWCNT preparations have been presented in Figure 2.As evident from the SEM micrograph, the final conjugate(viz., HA-EDBE-MWCNT-DOX) possessed average lengthin the range 0.3−0.6 μm and diameter ranging from 50 to 60 nm.Consistent with SEM observation, TEM image of HA-EDBE-MWCNT-DOX reveals well-individualized structure withaverage diameter ranging from 0.5 ± 0.1 μm and diameter rang-ing from 20 to 30 nm. It should be mentioned in this regardthat the diameters of HA-functionalized MWCNTs obtainedfrom TEM are, however, smaller than the diameter obtainedfrom SEM investigations. The result is justified because organiccoatings or functionalizations adhered with solid supports likeCNTs are hardly discernible by TEM. As determined by DLSstudies, HA-MWCNTs present in course of the study werehighly stable in aqueous medium. Their stable nature wasmanifested in their hydrodynamic (HD) size, polydispersityindex (PDI), and zeta potential values, which remainedunaffected even after six months from their date of preparation(Supporting Information, Table S2). Exposure of these func-tionalized nanotubes to various culture media (DMEM, RPMI,etc.) in the presence of serum revealed no significantdeterioration in hydrodynamic sizes and surface charge up to96 h of incubation (data not shown), conferring their suitabilityfor in vitro and in vivo applications.

Table 1. Particle Characteristics of Functionalized MWCNTsa

f- MWCNTs SEM features TEM featuresDLS size(nm) PDI

zeta potential(mV)

Oxidized MWCNTs Length 0.3−0.8 μm, Diameter 20−30 nm Length 0.3−0.8 μm, Diameter 20−30 nm 172.8 ± 4.2 0.421 ± 0.052 −57.5 ± 2.7EDBE-MWCNTs Length 0.3−0.8 μm, Diameter 40−50 nm Length 0.3−0.8 μm, Diameter 20−30 nm 225.9 ± 2.7 0.437 ± 0.034 +9 ± 0.6HA-PEG-MWCNTs Length 0.3−0.8 μm, Diameter 60−80 nm Length 0.3−0.8 μm, Diameter 20−30 nm 451.3 ± 8.7 0.310 ± 0.023 −42 ± 4aAll values unless otherwise stated are expressed as mean ± SD (n = 6).

Figure 1. Schematic representation of functionalization of MWCNTs with multiple bioactives.

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DOX Release from CNTs. DOX was loaded to CNTs exploit-ing the supramolecular π−π stacking interactions between drugand nanotubes (see Supporting Information, Figure S4, TableS3 for details). DOX release from HA-MWCNTs was checkedat both pH 5.5 and 7.4 to mimic the tumor microenvironmentand normal physiological milieu, respectively (Figure 3). At pH

7.4, only 14.26% of the entrapped drug was found to bereleased from the surface of MWCNTs after 32 h of exposureto the release medium. As the pH of the release medium waslowered to 5.5, around 36.50% DOX was found to be releasedfrom HA-MWCNTs just within 24 h of incubation. This parti-cular release profile is beneficial from the standpoint of cancertargeting, since cancerous tissues have a slightly acidic pH withhigh enzymatic activity. This pH-dependent release behaviorsuggests that DOX will be released more in acidic tumor sitesthan in the normal tissues. Once the particles are internalizedinside the cells through HR-mediated endocytosis, the de-creased pH values are supposed to induce further accelerateddrug release inside the acidic tumor endosomes. At this low pH,the hydrophilicity of DOX increases due to the protonation ofNH2 group native to its structure. The increased hydrophilicityaids in overcoming the π−π stacking force while facilitating therelease of DOX from MWCNTs. Since the present formulation

was meant for intravenous administration, the release profile ofDOX from nanotubes was also repeated in the presence ofserum. Interestingly, only 15−17% DOX was detached fromthe nanotubes after 48 h of incubation (Figure 3). These resultsindicated that the functionalized nanotubes prepared in thisstudy exhibited good stability in physiological solution con-taining various salts and proteins.

In Vitro Cellular Uptake, Cytotoxicity, and ApoptosisStudies. Intracellular Uptake and Colocalization Study. Celluptake studies were performed in human nonsmall cell lungadenocarcinoma (A549) cell lines, known to overexpress theCD44 receptors specific for HA.19 To apprehend whether themain hyaluronate receptor (HR) is involved in the uptake ofthe HA-conjugated MWCNTs, A549 cells were incubated withHA-MWCNTs in the absence and presence of HA. Whileblockage of HR hardly altered the internalization of HAdeprived, EDBE-MWCNTs amine-functionalized, the uptake ofHA-functionalized CNTs was significantly suppressed (Sup-porting Information Figure S5). These results supported thatHA-MWCNTs were internalized by A549 cells through CD44receptor mediated endocytosis. It seems that endocytosis is notinvolved during translocation of EDBE into the cells, since theirinternalization was not affected by complete blockage ofendocytic pathway either by low temperature or by sodiumazide (data not shown). The results, however, are in line withthe reported pathway of nanotube uptake by passive diffusion.20

To further understand the surface-chemistry dependence off-MWCNTs on their intracellular trafficking behavior, A549 cellswere incubated with various f-MWCNTs and their colocaliza-tion with various cell organelles, viz., lysosomes, mitochondria,was studied using confocal laser scanning microscopy (CLSM).For lysosome and mitochondrial colocalization experiments,f-MWCNTs were covalently tagged with AF-647 (indicated asgray fluorescence), whereas lysosomes and mitochondria werestained with Neutral Red (NR, red fluorescence) andRhodamine 123 (Rh123, green fluorescence), respectively.The degree of overlap between AF-647 labeled CNTs and anorganelle-specific fluorescence dye was measured in terms ofPearson’s correlation coefficient (r). A colocalization coefficientclose to or greater than 0.5 (r ≥ 0.5) was indicative of goodcolocalization. As evident from the line display of AF-647labeled f-MWCNTs and NR stained lysosomes (Figure 4A),

Figure 2. (a−c) Scanning electron micrographs and (d−f) transmission electron micrographs of 3 h oxidized MWCNTs, EDBE-MWCNTs, andHA-EDBE-MWCNTs, respectively.

Figure 3. In vitro release profile of DOX from HA-MWCNTs at pH7.4 (physiological pH), serum, and pH 5.5 (tumor pH).

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following their intracellular uptake, HA-MWCNTs rapidly trans-located into the lysosome, whereas their nontargeted counter-parts were mainly distributed within the cellular cytoplasm. Asdetermined from the scattered plot (Figure 4B), the Pearson’scolocalization coefficients (r) of AF-647 and NR fluorescence forEDBE-MWCNTs and HA-MWCNTs were determined to be 0.2and 0.6, respectively. These values revalidate the results of linedisplay in which HA-MWCNTs was shown to present appreci-able colocalization with cellular lysosomes. In this context, it maybe noted that the colocalization determined in the entire field ofview (scatter plot) cannot precisely discriminate the fluorescenceassociated with cell surface adhered nanotubes from thosepresent intracellularly. To confirm the colocalization, line plotanalysis (Figure 4C) was performed that measures variationsin fluorescence intensity along a line. Yet again, the line plots

determine variations in fluorescence intensity, just in onedimension. To revalidate the quantitative colocalization results,box-plot analyses were performed (SI Figure S6), which mea-sure the changes in fluorescence intensity in two dimensions,that is, X and Y. Consistent with the results of scattered plotanalysis, both line and box colocalization analysis establishedthat HA conjugated MWCNTs exhibit good colocalization withthe lysosomes. Binding of HA-MWCNTs to HR possibly leadsto formation of endocytic vesicles,21 which, in turn, favoredtheir intracellular trafficking to lysosomes. This colocalizationprofile is beneficial from the standpoint of accelerated DOXrelease under lysosomal conditions. As already discussed, theacidic pH of lysosomes will aid in the protonation of NH2

group native to the structure of DOX and trigger drug releasefrom CNTs. We also observed some fluorescence emissions of

Figure 4. (A) Representative line display of A549 cells incubated with (a−d) EDBE-MWCNTs and (e−h) HA-MWCNTs. (a,e) indicates NRfluorescence; (b,d,f,h) AF-647 fluorescence and (c,g) Rh123 fluorescence. (B) Representative line plot of A549 cells incubated with (a,b) EDBE-MWCNTs and (c,d) HA-MWCNTs. (a,c) indicates line plot of AF-647 labeled CNTs and NR stained lysosomes; (b,d) line plot of AF-647 labeledCNTs and Rh123 stained mitochondria. (C) Representative scatter plot of A549 cells incubated with (a,b) EDBE-MWCNTs and (c,d) HA-MWCNTs. (a,c) indicates scatter plot of AF-647 and NR fluorescence; (b,d) scatter plot of AF-647 and Rh123 fluorescence.

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both EDBE-MWCNTs and HA-MWCNTs in the mitochon-dria. As determined from scattered plot analysis, Pearson’s colo-calization coefficient (r) of AF-647 and Rh123 fluorescence wasapproximately 0.5 in both cases. Because mitochondrial mem-brane is negatively charged, the mitochondrial colocalization ofEDBE MWCNTs with surface-pendant amine groups is not im-plausible and is also consistent with the proposed transmem-branal pathway of mitochondrial colocalization of PL-PEG-SWCNTs by Zhou et al. However, colocalization of negativelycharged HA-MWCNTs with mitochondria is a bit paradoxical.Although the mechanism of mitochondrial localization of HA-MWCNTs is not fully understood, it is quite feasible that, fol-lowing their internalization into the cells, HA-MWCNTs aretranslocated into lysosomes via endosomal pathway. There is a fairchance that the low pH and nonspecific proteases inside thelysosomal compartment will accelarate the hydrolysis of the amidebond between HA and EDBE so that a part of the surface aminegroups is rejuvenated to interact with the negatively charged mito-chondrial membrane. As suggested by some earlier reports, wecannot also disregard the possibility of electrostatic interactionbetween the negatively charged functional groups of the glyco-saminoglycan backbone of HA and positively charged residues(viz., lys267, lys270, and arg 411) of cytochrome P450scc andadrenodoxin, some of the very well-known redox proteins involvedin the electron transfer chain of the inner mitochondrial mem-brane.22 However, without further studies, it is difficult to makeany precise comment on the underlying mechanism that trans-locates HA-conjugated CNTs to mitochondria. In this regard, itmay also be noted that Rh123 exhibits green emission, whereasthe dye NR shows red emission. Because of the shorter wave-length of green emission, the emitted fluorescence is somewhatscattered or diffused, which might increase the apparent colocaliza-tion to some extent. Whatever the case might be, from data inhand, it is confirmed that HA-CNTs enter the cells through CD44receptor mediated endocytosis and translocate into the lysosomes.Cytotoxicity Study. To further check the feasibility of DOX

release inside the target cells, A549 cells were cultured with freeDOX as well as DOX loaded MWCNT-EDBE-HA. DOX-deprived MWCNT-EDBE-NH2 and MWCNT-EDBE-HA weretaken as a control. While DOX-deprived MWCNTs led tonegligible reduction in cellular viability (EC-50≫ 100 μg/mL),both free DOX and HA-MWCNT-DOX exhibited dose-dependent reduction in cellular viability (Figure 5). At very

low concentration of DOX (0.001−0.01 μg/mL), insignificantdifferences were noted between the cytotoxicity of free DOXand DOX-loaded CNTs. Above this concentration (≥0.1 μg/mL), DOX delivered through CNTs always exerted highercytotoxicity than the free drug in equivalent concentrations.The IC50 of free DOX and HA-CNT-DOX were calculated tobe 2.117 and 0.8688 μg/mL, respectively. These results indicatethat our formulation is approximately 2.4 times more cytotoxicthan the free drug. These observations suggest that intracellularconcentration of DOX delivered through MWCNTs is higherthan that of free DOX. The hypothesis may be rationalized, ifwe consider the intracellular trafficking behavior of MWCNT-HA-DOX. As evident from the results of cell uptake andintracellular trafficking, HA-MWCNTs exhibited appreciablecolocalization with the lysosomes. This selective localizationpossibly augmented the release of free DOX from MWCNTs.In order to confirm that CNT-encapsulated DOX is specificallyreleased in the lysosomes, A549 cells were incubated withHA-MWCNT-DOX in the absence and presence of ammo-nium chloride, and cytotoxicity was determined in both cases.Notably, a marked reduction in cytotoxicity was observed whenthe cells were pretreated with NH4Cl. These results suggestthat addition of NH4Cl leads to accumulation of ammoniumions in the lysosomes while increasing their pH.23 As DOXrelease from nanotubes is augmented under low pH conditions,an increase in lysosomal pH condition disfavors drug releasefrom CNTs. Overall, these observations suggest that encap-sulation of DOX within HA-MWCNTs not only enhance theirintracellular uptake but also traffick the therapeutic vector tothe lysosomes wherein the drug detaches from the nanotubesurface, diffuses out into the cytosol, and subsequently trans-locates to its site of action, viz., nucleus.

Apoptosis Study. Reduction in cellular viability is normallyassociated with concomitant increase in the number of apopto-tic cells. In order to substantiate whether our formulation caninduce cell death through the initiation of apoptosis, A549 cellswere incubated in PBS with 1 μg/mL of free DOX and HA-CNTs containing equivalent amounts of free drug. The varioushallmarks of apoptosis, viz., phosphatidyl serine (PS) external-ization, mitochondrial depolarization, nuclear condensation,and DNA fragmentation, were studied qualitatively as well asquantitatively using Annexinn V (AV)-Cy3 apoptosis detectionkit, Rh123 staining, propidium iodide (PI) staining, TUNEL,and DPA assays. While no hallmarks of apoptosis were detectedwith control, cells incubated with free drug as well as drug-loaded CNTs exhibited prominent features of early as wellas late apoptosis including PS externalized cells, condensedand fragmented nuclei, as well as depolarized mitochondria(Figure S7). As evident from Figure.6, cells incubated with freeDOX and HA-CNT-DOX presented 60 ± 5.8% and 75.1 ±8.7% PS externalized (AV + ve) cells, and the difference wasstatistically insignificant. Notably, cells incubated with HA-MWCNT-DOX presented a significantly higher number of cellsin late apoptosis (78 ± 7.7) when compared with the free drug(55 ± 6.8). Further cells incubated with HA-MWCNTs dis-played higher percentage of fragmented nuclei and DNA, ascompared to free DOX. These results suggest that DOX deli-vered through CNTs induced apoptosis more effectively thanthe free drug.

In Vivo Studies. Biodistribution Studies after Radio-labeling of f-MWCNTs. Radiolabeling Studies. In order tomonitor the blood clearance kinetics and organ distributionbehavior of our NPs in vivo, all the formulations were

Figure 5. Dose-dependent cytotoxicity of A549 cells treated with freeDOX and MWCNT-HA-DOX.

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radiolabeled with 99mTc, using stannous chloride as thereducing agent. 99mTc was chosen for the present study becauseof its ease of availability, cost effectiveness, and low radiationdose. The half-life of Tc is only 6 h, which ensures less radiationburden when compared to other radionuclides such as I128

presenting a longer half-life (∼60 days). HA-functionalizedMWCNTs, developed in the course of the present study, weresuccessfully labeled with Tc-99m with more than 98% labelingefficiency.24,25 The concentration of SnCl2 had a significantinfluence on the labeling yield. At low concentration of thestannous chloride, Tc was not properly reduced, but at higherconcentration, it led to the formation of a reduced, hydrolyzed(RH) form of Tc. Therefore, to optimize the labeling pro-cedure, the concentration of SnCl2 was varied from 25 to300 μg, keeping other parameters constant. The maximumlabeling efficiency for all the formulations was obtained at100 μg of SnCl2 concentration.

25 The in vitro stability of thelabeled formulations was evaluated in PBS (pH = 7.4) as well asserum. All the formulations showed excellent stability in vitro.The formulations were approximately 91−94% stable after 24 h(Table S4, Supporting Information).Organ Distribution Studies. Biodistribution of free DOX as

well as f-MWCNTs was assessed in EAT bearing mice model, awell-established animal model for CD44 receptor overexpress-ing tumors. We observed that the organ distribution profile ofDOX-loaded CNTs insignificantly differed from those deprivedof DOX (data not shown), which suggests that biodistributionof CNTs in vivo critically depends on their surface functionalityand not on the encapsulated cytotoxin. In other words, bio-distribution of drug-deprived f-MWCNTs may be consideredequivalent to the biodistribution of their drug-loaded counter-parts. The percentage injected dose of free DOX, EDBE-MWCNTs, and HA-EDBE-MWCNTs per gram (% ID/g) oftissue in different organs, at different time points, has beenpresented in Table 2−4. As evident from organ distributiondata, not much difference could be chalked out among the dis-tribution and accumulation of free drug, MWCNT-EDBE-DOX, and MWCNT-EDBE-HA-DOX in heart, kidney,stomach, and intestine. Notably, significant differences wereobserved in the distribution profile of free DOX and DOXloaded functionalized MWCNTs in the MPS organs, viz., liver,spleen, and lungs. In liver, the % ID/g for MWCNT-EDBE-DOX and MWCNT-EDBE-HA-DOX were observed to be

21.1 ± 1.48 and 17 ± 1, respectively, after 0.5 h, while % ID/gfor free DOX was calculated to be only 10.5 ± 1.52. Likewise,in spleen, the % ID/g for MWCNT-EDBE-DOX (10 ± 0.2)and MWCNT-EDBE-HA-DOX (7.85 ± 0.25) (after 0.5 h) werefound to be significantly higher than free DOX (3.1 ± 0.48).Similarly, the % ID/g values in lungs after 0.5 h were detectedto be 5.8 ± 1.03, 10.08 ± 0.24, and 22.65 ± 0.55 for free DOX,MWCNT-EDBE-DOX, and MWCNT-EDBE-HA-DOX, re-spectively. The results are justified and pretty consistent withthe observations of Qu et al.26 According to their report, bothagglomerated and well-suspended MWCNTs are taken up byMPS organs. However, MWCNTs with higher degrees ofagglomeration are retained in lungs and later in the liver formonths, while the well-dispersed ones formed fewer aggregates

Figure 6. Evaluation of HA-MWCNT-DOX and free DOX inducedapoptosis in A549 cell line: Quantitative determination of early andlater apoptotic cells, condensed nuclei and fragmented nuclei/DNAusing Annexin V assay, TUNEL assay, PI staining, and DPAcolorimetric assay, respectively. All data are expressed as mean ± SD(***p < 0.001, **p < 0.01, *p < 0.05).

Table 2. In Vivo Biodistribution Profile of Free DOX

% ID/g recovered after

organ or tissue 0.5 h 2 h 4 h 24 h

blood 0.91 ± 0.12 0.66 ± 0.11 0.52 ± 0.13 0.15 ± 0.04heart 0.49 ± 0.11 0.32 ± 0.08 0.23 ± 0.06 0.06 ± 0.02lungs 5.8 ± 1.03 4.3 ± 0.97 2.1 ± 0.71 0.32 ± 0.07liver 10.5 ± 1.52 7.04 ± 1.5 6.9 ± 1.32 2.3 ± 0.69spleen 2.1 ± 0.48 2.13 ± 0.4 1.65 ± 0.39 0.63 ± 0.14kidney 2.9 ± 0.8 2.01 ± 0.72 1.97 ± 0.41 0.56 ± 0.19stomach 0.53 ± 0.12 0.48 ± 0.14 0.42 ± 0.12 0.07 ± 0.01intestines 0.45 ± 0.13 0.31 ± 0.06 0.22 ± 0.06 0.05 ± 0.01tumor 0.19 ± 0.04 0.39 ± 0.08 0.37 ± 0.1 0.11 ± 0.02muscle 0.17 ± 0.03 0.15 ± 0.03 0.11 ± 0.02 0.06 ± 0.01tumor/muscle 1.11 2.6 2.36 1.83

Table 3. In Bivo Biodistribution Profile of EDBE-MWCNTs

% ID/g recovered after

organ or tissue 0.5 h 2 h 4 h 24 h

blood 0.79 ± 0.03 0.65 ± 0.03 0.45 ± 0.02 0.11 ± 0.02heart 0.29 ± 0.02 0.17 ± 0.01 0.14 ± 0.02 0.08 ± 0.01lungs 22.65 ± 0.55 19.2 ± 0.4 15.8 ± 0.7 5.3 ± 0.4liver 21.1 ± 1.48 16.5 ± 0.7 14.21 ± 0.03 8.15 ± 0.65spleen 10 ± 0.2 6.91 ± 0.03 5.45 ± 0.45 4.15 ± 0.15kidney 1.63 ± 0.13 0.94 ± 0.04 0.68 ± 0.05 0.49 ± 0.05stomach 0.53 ± 0.03 0.49 ± 0.01 0.42 ± 0.02 0.26 ± 0.02intestines 0.66 ± 0.05 0.52 ± 0.02 0.32 ± 0.02 0.11 ± 0.01tumor 0.92 ± 0.06 1.09 ± 0.03 1.18 ± 0.12 0.50 ± 0.03muscle 0.21 ± 0.02 0.15 ± 0.02 0.12 ± 0.01 0.15 ± 0.02tumor/muscle 4.38 7.26 9.83 2.33

Table 4. In Vivo Biodistribution Profile of HA-EDBE-MWCNTs

% ID/g recovered after

organ or tissue 0.5 h 2 h 4 h 24 h

blood 0.62 ± 0.07 0.42 ± 0.01 0.27 ± 0.01 0.08 ± 0.01heart 0.22 ± 0.01 0.19 ± 0.01 0.13 ± 0.02 0.03 ± 0.01lungs 10.08 ± 0.24 7.9 ± 0.03 6.21 ± 0.02 2.12 ± 0.18liver 17 ± 1 14.6 ± 0.7 11.3 ± 0.3 7.95 ± 0.55spleen 7.85 ± 0.25 6.15 ± 0.35 4.95 ± 0.15 1.8 ± 0.1kidney 1.16 ± 0.04 0.86 ± 0.06 0.68 ± 0.03 0.29 ± 0.02stomach 0.66 ± 0.05 0.52 ± 0.03 0.41 ± 0.01 0.07 ± 0.01intestines 0.51 ± 0.01 0.29 ± 0.02 0.16 ± 0.01 0.08 ± 0.01tumor 1.06 ± 0.03 1.65 ± 0.15 2.5 ± 0.3 1.60 ± 0.23muscle 0.14 ± 0.02 0.16 ± 0.01 0.18 ± 0.01 0.15 ± 0.03tumor/muscle 7.57 10.31 12.8 10.6

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in lungs and liver, and seemed to be easily eliminated viaexcretion. In the present case, it was quite interesting to observethat % ID/g in liver, spleen, and lungs steadily decreased as afunction of time. Following 24 h of administration, the % ID/gvalues in liver were detected to be 2.3 ± 0.69, 5.3 ± 0.4, and3.12 ± 0.18 for free DOX, MWCNT-EDBE-DOX, andMWCNT-EDBE-HA-DOX, respectively. These data revalidatethe hypothesis that functionalized MWCNTs with shorterlength and high aqueous dispersibility exhibit minimum pro-pensity to accumulate in the liver and over the course of time,eliminated via urinary excretion or biliary pathway in the feces.Organ distribution data further reveal that free DOX wasgradually cleared away from the blood with time; after 24 h, the% ID/g in blood was only 0.15 ± 0.04. The results are quitejustified, because DOX, being a hydrophilic drug, is rapidlyeliminated from the body through urinary excretion. Carefulscrutiny of the organ distribution data shows that %ID/g inkidney for free DOX at any time point is always higher than theCNT-based formulations. As in the case of free DOX, bothMWCNT-EDBE, as well as its targeted counterpart, viz.,MWCNT-EDBE-HA, exhibited apparent clearance from bloodcirculation. However, both formulations, instead of beingeliminated from the body via excretory pathways, accumulatedin the tumor site. In the case of free DOX, the % ID/g in tumorafter 4 h was detected to be 0.38 ± 0.1% while % ID/g values inthe case of MWCNT-EDBE and MWCNT-EDBE-HA weredetermined to be 1.18 ± 0.56 and 2.5 ± 0.3, respectively. Theformulations were able to retain in these sites even after 24 h ofadministration. After 24 h, the tumor to muscle ratio for freeDOX, MWCNT-EDBE-DOX, and MWCNT-EDBE-HA-DOXwere calculated to be 1.83, 3.33, and 10.6, respectively, implyingthat tumor-specific accumulation of HA-targeted MWCNTs isaround 5.79 and 3.18 times higher as compared to free DOXand HA nontargeted, EDBE functionalized MWCNTs.Tumor Growth Inhibition Study. In vivo tumor growth

inhibition study was carried out in DMBA induced Sprague−Dawley female rats, injected intravenously with a single dose of5 mg/kg of plain DOX and MWCNTs in equivalent concen-tration of the free drug. Figure 7 presents the tumor-growth

inhibition profile of rats treated with various MWCNTpreparations. A single i.v. administration of HA-PEG-

MWCNT-DOX led to a significant reduction in tumor burdenwithin 24 h of administration. The minimized tumor volume inrats after 10 days of treatment with HA-EDBE-MWCNT-DOXwas approximately 18, 17, 5, and 3 times less as compared torats exposed to saline, drug-deprived HA-EDBE-MWCNTs,free DOX, and EDBE-MWCNT-DOX, respectively. Further-more, animals treated with free DOX at the dose level specifiedinduced significant signs of toxicity, i.e., pronounced weightloss, that was not observed for any of the MWCNT treatedgroups. In fact, rats treated with free DOX appeared somewhatsluggish and congregated at the corner of the cage just after day1 of treatment. Conversely, in the case of CNT treatment, allthe animals appeared lively throughout the study, and nosubstantial weight loss was detected. The higher antitumorefficacy of HA-EDBE-MWCNT-DOX may be attributed to itshigh tumor-specific accumulation, facilitated by the followingfactors: (i) enhanced permeability and retention (EPR) effect,mediated by the short, hydrophilic, PEG-like chain adheredto the surface of CNTs (passive targeting); (ii) selective up-take of functionalized nanotubes via HR mediated endocyto-sis (active targeting); and (iii) their high intracellular pene-trability. It should, however, be noted that non-HA conjugatedEDBE-MWCNTs also displayed antitumor activity to anappreciable extent by virtue of EPR/stealth effect. The resultsof tumor-growth inhibition studies are in very good agreementwith our organ-distribution data, according to which HA-conjugated MWCNTs exhibited around 5.79 and 3.18 timeshigher accumulation in the tumor site as compared to free drugand its nontargeted counterpart, even after 24 h of adminis-tration. Our findings are also consistent with the reports ofYadav et al. according to which antitumor efficacy of DOXloaded in PCL-PEG and PLGA-PEG core−corona nanoparti-cles were significantly enhanced on HA conjugation.24,27 In thecase of rats exposed to DOX deprived HA-EDBE-MWCNTs, itwas quite interesting to observe that, up to 4 days post-treat-ment, the percentage increase in tumor volume was con-siderably lower as compared to the control. Following 6 dayspost-treatment, the increased tumor volume (%) of HA-EDBE-MWCNTs was almost comparable to that of the control group.Remarkably, at this point, no significant increase in tumorvolume was observed for the MWCNT treated group, while asteady increase was still observed for the control group. Theobservations can be rationalized in light of a recent report byMeng et al.,28 according to which, subcutaneous (s.c.) injectionof oxidized MWCNTs could effectively enhance the immunityin mice leading to certain beneficial effects such as tumor-growth inhibition. Experimental results showed that CNT-induced significant activation of the complement system pro-moted production of inflammatory cytokines and stimulatedsubsequent phagocytosis and activation of macrophages. Collec-tively, all these responses are believed to increase the generalactivity of the host immune system and, in turn, synergized thetumor growth inhibition process.

Toxicity Studies in Rats and Mice. Drug-associated cardio-toxicity is a severe limitation of DOX therapy. Hence, it wasnecessary to evaluate and compare the cardiotoxicity offree DOX as well as DOX loaded f-MWCNTs. Subsequently,the various cardiotoxicity markers such as lactate dehydrogenase(LDH), cytokinin MB (CK-MB), and aspartate trans-aminase (AST) level in plasma and superoxide dismutase(SOD) activity in heart homogenate were determined. Anincrease in LDH, CK-MB, or AST level and decrease inSOD activity indicates cardiotoxicity. As evident from the

Figure 7. Tumor growth inhibition profile of SD rats intravenouslyadministered with free DOX and various functionalized MWCNTpreparations loaded with equivalent amount of the free drug.

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various cardiotoxicity marker levels, both MWCNT-EDBE-DOX and MWCNT-EDBE-HA-DOX showed markeddepreciation in cardiotoxicity as compared to free DOX.It is clear from Figure 8a,b that, after 10 days of treatment,LDH level in heart tissue and CK-MB level in the plasmawere significantly increased in the animal groups treatedwith free DOX. Conversely, LDH and CK-MB levels ofvarious CNT-treated groups showed insignificant differ-ences from the control group. Concomitantly, SOD levelsin the heart homogenate decreased in animals treated withfree DOX, while both MWCNT-EDBE-DOX andMWCNT-EDBE-HA-DOX showed comparable SOD activ-ity with respect to control (Figure 8c). Additionally, theAST level of free DOX treated rats was significantlyelevated, indicative of myocardial infarction, whereas ratstreated with functionalized CNTs showed insignificantdifferences from the control (Figure 8d). As in the casewith rats, DOX delivered through HA-MWCNTs wasdevoid of any detectable cardiotoxity, hepatotoxicity, andnephrotoxicity in mice (see Supporting Information FiguresS8, S9). These results overall suggest that the deliveryproperty of MWCNTs is effectively combined with thetargeting property of HA to augment the anticancer activityof DOX against hyaluronate receptor bearing tumor cells,while reducing the toxicity associated with both drug andthe carrier system.

■ CONCLUSIONS

In conclusion, the delivery property of MWCNTs has beensuccessfully blended with the targeting property of HA toaugment the antitumor activity of DOX against HR over-expressing cancer cells while reducing the drug-associated cardio-toxicity. The therapeutic conjugate was further labeled with anear-infrared fluorescent dye, Alexa-Fluor-647 (AF-647), andradiotracer Technetium-99m to track the intracellular traffickingand biodistribution of the conjugate via optical imaging andscintigraphic techniques. Studies related to the evaluation of long-term toxicity and fate of DOX-loaded, HA-tethered MWCNTsis currently underway in our laboratory. The results may bereported in due course. Overall, the carrier system, developed incourse of the present study, may expand the therapeutic transomfor a broad spectrum of anticancer drugs and considered forclinical applications.

■ ASSOCIATED CONTENT

*S Supporting InformationMethods of surface oxidation of pristine MWCNTs, colori-metric assay for quantification of surface amine density, in vitrodrug loading studies, radiolabeling studies, in vivo stabilitystudies, toxicity studies, surface chemistry analysis off-MWCNTs using FTIR and HRMAS NMR spectroscopy,quantification of functional molecules associated with

Figure 8. Evaluation of various cardiotoxicity parameters: (a) LDH; (b) CK-MB; (c) SOD; and (d) AST in tumor bearing rats treated with freeDOX/DOX-loaded MWCNTs) [a* w.r.t. control (***p < 0.001, **p < 0.01, *p < 0.05); ULN = upper limit of normal].

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MWCNTs via TG analysis, stability of HA-MWCNTs inaqueous medium, DOX loading on HA-MWCNTs, results ofcompetitive inhibition and apoptosis study, in vitro and in vivostability of radiolabeled formulations and results of in vivotoxicity in mice. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*Tel.: +91172-2292055, Fax: +91172-2214692 E-mail addresses:sanyogjain@niper.ac.in, sanyogjain@rediffmail.com.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The authors are thankful to Indian Council of MedicalResearch (ICMR) and Department of Science & Technology(DST), Government of India, New Delhi, for financial support.M.D. and R.P.S. are grateful to Department of Science andTechnology (DST), GOI, New Delhi, for providing post-doctoral and senior research fellowships, respectively. DirectorNIPER and Director INMAS are duly acknowledged forproviding the necessary infrastructure and facilities. Theauthors are grateful to Dr. Anil K. Mishra and his group,DCRS, INMAS for providing necessary facilities for radio-labeling and scintigraphy studies. Thanks are due to Dr. Ravi S.Amarpati, SAIF, Central Drug Research institute (CDRI,Lucknow) for assistance with HRMAS analysis. Technicalassistance of Mr. Dinesh Singh Chauhan and Mr. RahulMahajan is also acknowledged.

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