Intranuclear Drug Delivery and Effective in Vivo Cancer Therapy viaEstradiol−PEG-Appended Multiwalled Carbon NanotubesManasmita Das, Raman Preet Singh, Satyajit R. Datir, and Sanyog Jain*

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

*S Supporting Information

ABSTRACT: Cancer cell-selective, nuclear targeting isexpected to enhance the therapeutic efficacy of a myriad ofantineoplastic drugs, particularly those whose pharmacody-namic site of action is the nucleus. In this study, a steroid-macromolecular bioconjugate based on PEG-linked 17β-Estradiol (E2) was appended to intrinsically cell-penetrablemultiwalled carbon nanotubes (MWCNTs) for intranucleardrug delivery and effective breast cancer treatment, both invitro and in vivo. Taking Doxorubicin (DOX) as a modelanticancer agent, we tried to elucidate how E2 appendageinfluences the cell internalization, intracellular trafficking, andantitumor efficacy of the supramolecularly complexed drug.We observed that the combination of DOX with E2-PEG-MWCNTs not only facilitated nuclear targeting through an estrogen receptor (ER)-mediated pathway but also deciphered to asynergistic anticancer response in vivo. The antitumor efficacy of DOX@E2-PEG-MWCNTs in chemically breast cancer-inducedfemale rats was approximately 18, 17, 5, and 2 times higher compared to the groups exposed to saline, drug-deprived E2-PEG-MWCNTs, free DOX, and DOX@m-PEG-MWCNTs, respectively. While free DOX treatment induced severe cardiotoxicity inanimals, animals treated with DOX@m-PEG-MWCNTs and DOX@E2-PEG-MWCNTs were devoid of any perceivablecardiotoxicity, hepatotoxicity, and nephrotoxicity. To the best of our knowledge, this is the first instance in which cancer cell-selective, intranuclear drug delivery, and, subsequently, effective in vivo breast cancer therapy has been achieved using estrogen-appended MWCNTs as the molecular transporter.

KEYWORDS: cancer, intranuclear drug delivery, estrogen, carbon nanotubes, antitumor efficacy

1. INTRODUCTION

Estrogen hormones, in particular, 17β-estradiol (E2), have beenidentified as one of the most vital hormones regulating thedevelopment and maintenance of the female reproductivesystem and secondary sex characteristics.1−3 Binding of E2 withestrogen receptors (ER) induces conformational changes andrelease of molecular chaperone (Hsp 90, Hsp 70, cyclophilin,and p23) from the receptors,4 which allows them to conscriptthe cofactors necessary for transcription of various genescommonly upregulated in malignant cells (e.g., transforminggrowth factor alpha, c-myc, or cathepsin D). As evident from anextensive literature survey, hormone receptors like ERs andprogesterone receptors are overexpressed in 70−80% of allbreast cancers. The upregulation of ERs in cancerous cellsrelative to normal cells can be effectively harnessed for thedevelopment of a targeted therapy against various hormonesensitive cancers.5,6 ER-α is known to localize in both nucleusas well as plasma membrane, mediating estrogen-dependent,genomic, and nongenomic signaling.4,7−10 Subsequently,conjugation of estrogen hormones with any pharmaceuticallyactive component (either free or carrier bound) maysimultaneously facilitate cellular and intracellular, organelle-

specific (nuclear) targeting in such receptor overexpressedcancer cells.A large number of first line chemotherapeutic medications,

including doxorubicin (DOX) and cisplatin (CDDP), exerttheir pharmacodynamic effects in nuclei by intercalating withDNA base pairs, thereby inhibiting cell growth/proliferation.11

Unfortunately, the transport of these anticancer drugs fromplasma membrane to nucleus is not very well-characterized. Infact, transport of drugs to nuclei have been found to be ratherdifficult, and even if it could happen, it is considered to benonspecific and passive. Additionally, drug-resistant cancer cellshave many intracellular drug-resistance mechanisms that avertthe access of anticancer agents to the nucleus. Consequently,only a small percentage of the administered dose can bedelivered into the cytosol and finally reach the nucleus.9 Overthe past years, a number of ER-targeted bioconjugates havebeen prepared by coupling estrogens with a myriad of cytotoxic

Received: April 21, 2013Revised: July 7, 2013Accepted: August 1, 2013

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drugs, including taxol,12 nitrogen mustards,13 genototoxins,14

geldanamycin,15 porphyrins,9 and enediynes.16 Unfortunately,the ER-binding affinities of these conjugates were largelycompromised due in part to the modification of the parentestradiol molecule and addition of appendages of varyingchemical nature and steric bulk. Subsequently, the desireddegree of accumulation of the conjugate selectively into thetumor often remains unachieved. On the other side, a myriad ofnano drug delivery systems have been developed to targetcancer cells, but only limited recent reports have focused on thefeasibility of nuclear targeting using nanoparticles.17,18 In thosefew instances too, the investigations were restricted to mere invitro evaluations so that it is not possible to predict whetherdirect intranuclear release of therapeutic drugs in vitro could bedeciphered into a comparable therapeutic response in vivo. Atthe same time, a few recent reports have embarked on the

feasibility of targeted cancer therapy using steroids anchoredliposomes/gold nanoparticles as the therapeutic vectors.19−21

However, from the reported studies, it is not clear whethercombination of an estrogen/estrogen antagonist with thenanocarrier could deliver the drug directly into the nucleus.We reasoned that tumor-specific accumulation as well asintranuclear drug delivery could be significantly enhanced, if thedrug molecule of interest is loaded into an E2-anchorednanocarrier that has a natural propensity for intracellularpenetration in addition to energy-dependent internalization vialigand−receptor affinity interactions. Amidst the innumerablenanocarriers that have elicited promise with regard to targeteddrug delivery or imaging, functionalized carbon nanotubes (f-CNTs) have sparkled phenomenal interest.22−25 The uniquephysicochemical and structural properties of these nanocarriersenable multiple diagnostic and therapeutic moieties to be

Figure 1. A schematic illustration for the synthesis of an estradiol−PEG-MWCNT conjugate.

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integrated on the same nanotube platform, while their naturalshape facilitates their noninvasive penetration across biologicalbarriers. In one of our recently published reports, we haveembarked on the feasibility of modulating the intracellulartrafficking and therapeutic performance of drug-loaded carbonnanotubes by intelligent manipulation of their surfacechemistry.26 In this study itself, we championed the idea ofnuclear-specific drug targeting using estradiol-appended multi-walled carbon nanotubes (MWCNTs) as the therapeuticvector. However, our previous study was just a proof-of-concept; we did not provide direct evidence of intranucleardrug release, and also our investigation was limited to mere invitro evaluations. With motivation from our previous work, wesought to design a novel, nuclear-targeted, CNT-basedbioconjugate that can potentially augment the therapeuticefficacy of its associated cytotoxin in vivo while minimizingdrug-associated toxicities. In line with this idea, a novel,poly(ethylene glycol)-linked E2 derivative viz. 17β-estradiol-hemisuccinyl-poly(ethylene glycol)-amine (E2-PEG) was syn-thesized and covalently conjugated to carboxylated MWCNTs.As established in earlier studies from our group as well asothers’, we appended E2 and CNTs to the distal ends of a PEGspacer because such a surface design effectively (i) minimizesnonspecific sequestration of CNTs by the mononuclearphagocytic system (viz. liver and spleen) and (ii) increasescarrier-localization to target tumor site via enhanced perme-ation and retention (EPR) effect (passive targeting).27,28

Taking DOX as a model anticancer agent, we tried to elucidatehow E2 modification influences the cancer targetability,intracellular trafficking, and anticancer efficacy of the conjugateboth in vitro and in vivo. We observed that combination ofDOX with E2-PEG-MWCNTs not only facilitated intranucleardrug delivery through an ER-mediated pathway but could beeffectively translated into a synergistic therapeutic response invivo while reducing drug-associated cardiotoxicity. To ourknowledge, this is the first example in which MWCNTs havebeen covalently tethered with a PEGylated E2 derivative andsuccessfully explored for cancer cell selective intranuclear drugdelivery, imaging, and effective therapy against breast cancer invivo.

2. MATERIALS AND METHODS2.1. Materials. Pristine (p) MWCNTs (purity >95%, length

1−5 μm, and diameter 20−30 nm) were procured fromNanovatech Pvt. Ltd., U.S. sulphuric acid, nitric acid (69−72%), disodium hydrogen phosphate, sodium acetate, sodiumbicarbonate, thionyl chloride, sodium lauryl sulfate, coppersulfate, and thiobarbituric acid were purchased from LobaChemie Pvt. Ltd., India. Doxorubicin was obtained as a giftsample from Sun Pharmaceuticals, India. PEG bisamine (Mw =3500) and methoxy-PEG were procured from JenKemTechnology. 17β-Estradiol (E2), succinic anhydride, dicyclo-hexyl carbodiimide (DCC), N-hydroxysuccinimide (NHS),neutral red (NR), rhodamine 123 (Rh123), 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), rhodamine B isothio-cyanate (RITC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium (MTT), and 7,12-dimethylbenz [α]anthracene (DMBA≥ 95% pure) were purchased from Sigma. All kits forbiochemical estimations were procured from Accurex, Bio-medical Ltd., Mumbai. Culture medium and serum wereprocured from PAA, Austria. Human lung carcinoma (A549),breast adenocarcinoma (MCF 7), and cervical cancer (HeLa)cells were obtained from the National Centre for Cell Sciences

(NCSS), Pune, India. Maleimide ester of Alexa-Fluor 647 (AF-647) was purchased from Invitrogen. All other chemicals/solvents were of analytical grade and procured from localsuppliers unless otherwise stated.

2.2. Methods. 2.2.1. Synthesis and Structural Elucidationof E2-PEG-MWCNT-Conjugate. The synthesis of E2-PEG-MWCNT-conjugate was accomplished in three steps asdepicted in Figure 1. Chemical structures of all synthesizedcompounds including final product and intermediates werepreliminarily analyzed by Fourier transform-infrared (FT-IR)spectroscopy and further authenticated via proton (1H) andcarbon (13C) nuclear magnetic resonance (NMR) spectrosco-py. The molecular mass of the E2-PEG derivative wasdetermined via matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectroscopy. The presenceof various functional groups on the surface of MWCNTs waspreliminarily studied through FT-IR, while fine-resolvedstructural characterization of surface-bound ligands was donethrough high-resolution magic angle spinning (HRMAS) NMRspectroscopy. Samples for HRMAS NMR experiments wereprepared by suspending 10 mg of each nanoparticle preparationin a 1:1 mixture of DMSO-d6:D2O (500 μL). HRMAS−NMRanalysis was carried out with a 400 MHz FT-NMRspectrometer (Avance 400) equipped with a 5 mm HRMASprobe.

2.2.1.1. Synthesis of Estradiol 17 β-Hemisuccinate.Estradiol 17 β-hemisuccinate was prepared by a simple, rapid,high yield, and relatively inexpensive procedure, earlier reportedby Yellin but with minor modifications.29 Briefly, 17β-estradiol(300 mg, 1.1 mmol) was dissolved in anhydrous benzene (10mL) and refluxed with approximately 5-fold molar excess ofsuccinic anhydride (550 mg, 5.5 mmol) in the presence ofpyridine (2 mL). The reflux was continued for approximately24 h, when E2 was no longer detectable by thin layerchromatography (TLC). Upon cooling to room temperature,excess succinic anhydride precipitated out from the solution,which was filtered off. The filtrate was concentrated underreduced pressure by rotary evaporation, following which, theresidue (E2-3, 17 disuccinate) was dissolved in methanol (10mL) and stirred overnight with an excess of sodium bicarbonate(1 g suspended in 10 mL of water) to complete selectivehydrolysis of the phenolic ester. Completeness of the reactionwas ensured by thin layer chromatography (TLC) using a 1:1(v/v) mixture of dichloromethane and methanol as the eluent.The reaction mixture was subjected to filtration to removeunreacted NaHCO3. After filtration of NaHCO3, water (10mL) was added to the reaction mixture. The alkaline solutionwas extracted 3 times with diethyl ether (10 mL × 3). Theaqueous phase was brought to pH 7 with 1 N HCl and thenpoured into a mixture of 0.1 N HCl and crushed ice. The whitecrystalline product that separated was removed by vacuumfiltration, washed repeatedly with water, and air dried. Thecrude E2-hemisuccinate was recrystallized from boiling benzene.Yield: 91.2%, white solid FTIR (ν, KBr pellets, cm−1): 3325(−OH carboxyl stretch), 2931, 2845, 2813 (−C−H, str), 1736(−O−CO, ester), 1610, 1510 (CH, aromatic str), 1220, 1170,1170 (C−O, −C−C), 785, 717 (−CH bend); 1H NMR (δ,DMSO-d6, ppm): 7.1−7.0 (d, E2C1H, 1H), 6.5−6.4 (d,E2C2H, 1H), 6.4−3.3 (d, E2C4H, 1H), 4.6−4.5 (t, E2C17H,1H), 3.8−3.6 (t, E2C20H, 2H), 3.6−3.4 (t, E2C21H, 2H), 2.7−2.6 (E2C6H, 2H), 2.5−2.4 (E2C7H, 2H), 2.3−2.2 (m, E2C16H,2H), 2.1−2.0 (m, E2C15H, 2H), 1.9−1.7 (m, E2C12H, 2H),1.6−1.5 (m, E2C15H, 2H), 1.4−1.0 (m, E2C8H, 2H), 0.8−0.7

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(m, E2C9H, 1H), 0.7−0.6 (s, E2C18H, 3H). Mass: 372 (M +H).2.2.1.2. PEGylation of E2-Hemisuccinate: Synthesis of E2-

PEG Derivative. The E2-hemisuccinate (42 mg, 0.1 mmol) indichlormethane (DCM, 2 mL), pyridine (2 mL), DCC (25 mg,0.12 mmol), NHS (15 mg, 1.3 mmol) were sequentially added.The resultant mixture was stirred for 1 h, following which PEG-bisamine (350 mg/0.1 mmol dissolved in 1 mL of DCM) wasadded. After 24 h, a finely suspended, white precipitate ofdicyclohyxyl urea (DCU) was envisaged in the reactionmixture, ensuring successful transformation of E2-hemisuccinateto the corresponding NHS ester. The precipitate was removedby filtration, following which the filtrate was added dropwise toice cold ether (30−40 mL) to precipitate the PEGylated E2derivative as a white semisolid. The crude product was air-dried,washed repeatedly with cold methanol to remove unreactedPEG-bisamine, and finally dried in vacuum. Yield: 71%. FTIR(νmax, KBr pellets cm

−1): 3430 (−OH stretch), 3010 (Ar C−H,str), 2918 (−CH stretch), 1756 (−O−CO, ester), 1680, 1626(CO, amide), 1456 (−N−H, bending), 1112, (CO, PEG); 1HNMR (δ, DMSO-d6, ppm): 8.9−8.8 (m, −CONHCH2, 1H),8.7−8.6 (d, dFdC-C2H, 1H), 8.2−8.1 (d, E2C1H, 1H), 8.0−7.9(d, E2C2H, 1H), 7.7−7.6 (d, E2C4H, 1H), 6.3−6.2 (t, E2C17H,1H), 6.0−5.9 (d, dFdC−C6H, 1H), 4.3−4.2 (d, dFdC−C5H,1H), 4.0−3.9 (d, dFdC−C4H, 1H), 3.9−3.2 (dFdC−C1H,C4H overlapped with b, −OCH2CH2), 2.7−2.6 (E26H), 2.6−2.5 (E2C7H), 2.4−2.3 (E2C12H);

13C NMR (δ, DMSO-d6,ppm): 210−200 (−CO−), 190−180 (R−CO−X, ester, amide),130−125 (Ar−C), 75−70 (−OCH2−CH2−O, PEG), 42−40(CH2NH−), 35−30 (R3C), 30−25 (3 °CCH2CH2CO), 25−20(R-CH2R), 15−10 (−CH3). Mass (MALDI-TOF): 3823 (M+).2.2.1.3. Functionalization of MWCNTs with PEGylated E2-

Hemisuccinate. Carboxyl-enriched oxidized MWCNTs wereprepared in accordance with our previous reports.30−32 Forfunctionalization of MWCNTs with E2, acid-oxidizedMWCNTs were converted to their corresponding acid-halidederivative by refluxing with thionyl chloride. Briefly, oxidizedMWCNTs (100 mg) were dispersed in THF (10 mL) viaultrasonication for 1 min. To the resultant dispersion, SOCl2(15−20 mL) was added, and the mixture was refluxed at 80 °Cfor 24 h.33 Thereafter, solvents were removed using rotavaporand the resultant acylated MWCNTs. As determined fromthermo-gravimetric analysis (TGA), carboxylic density on thesurface of MWCNTs was determined to be 0.0018 mmol/mgof MWCNTs. This value was necessary to calculate the exactamount of E2-PEG-NH2 that would be ideally required forcomplete interchange of surface carboxyl groups with thePEGylated steroid. On the basis of TGA results, around 3-foldmolar excess of E2-PEG-NH2, dissolved in anhydrous DMSO,was added to a suspension of acylated MWCNTs in a 5:1 (v/v)mixture of DMSO and pyridine under ice cold conditions. Thereaction mixture was left stirring for 24 h, following which thenanotubes were isolated by centrifugation of the supernatant.The pellet of functionalized MWCNTs was purified byrepeated washing with distilled water and acetone. Finally,the pellet was freeze-dried using an optimized freeze-dryingcycle, recently patented by our group.34 Lyophilized MWCNTswere used for further in vitro and in vivo studies. Yield: 80%(w/w).2.2.1.4. Functionalization of MWCNTs with m-PEG. For

preparation of PEGylated MWCNTs, carboxylated MWCNTs(100 mg) was acylated as described above and reacted with m-

PEG 3500 (in a 1.1 mol equiv of surface carboxyl density) for24 h to afford the PEGylated conjugate in the desired yield.Yield: 75% (w/w).

2.2.2. Size and Morphology of MWCNTs. Microstructuresof f-MWCNTs were analyzed using scanning electronmicroscopy (SEM, model S30400) and transmission electronmicroscopy (TEM, model FEI Tecnai G2).

2.2.3. DOX Loading and Release from MWCNTs. DOXloading on MWCNTs was consummated at pH 7.4 using thesame protocol described in our earlier reports.30 Briefly, E2-PEG-MWCNT and DOX were mixed in the ratio of 2:1 (w/w),and the resultant mixture was incubated at pH 7.4. TheMWCNT−drug mixture was kept in a shaker bath at 37 °C for24 h, following which, drug-loaded MWCNTs were separatedby centrifugation and absorbance of the supernatant wasmeasured at 480 nm.

2.3. In Vitro Cellular Uptake and Cytotoxicity Studies.Cell uptake studies were conducted in ER(+ve), A549, andMCF 7 cells.35 Briefly, cultured cells were exposed to various f-MWCNT-formulations (10 μg/mL) for 1 h. Cell internal-ization of MWCNTs was visualized via confocal microscopy(Olympus FV 1000 microscope) and quantified using standardspectrofluorimetry-based techniques. In order to apprehend theroles of ER with regards to intracellular uptake and subcellulartranslocation of the synthesized conjugate, A549 cells wereexposed to 50 μg/mL of different nanotube preparations in theabsence and presence of E2 (50 μg/mL).36 Cell internalizationwas monitored via confocal microscopy. Intracellular colocal-ization studies were performed by labeling lysosomes,mitochondria, and nuclei of A549/HeLa/MCF 7 cells withneutral red (NR), Rh123, and DAPI, respectively, as detailed inour earlier reports.37,38 In vitro cellular cytotoxicity waspreliminarily assessed by the conventional MTT assay. TheIC-50 values were determined using Graph Pad Prism software(version 6.03). Free DOX and DOX-CNT-induced cellularapoptosis/DNA fragmentation were quantified using terminaldeoxynucleotidyl transferase dUTP nick-end labeling(TUNEL) and diphenylamine (DPA) assays.

2.4. In Vivo Pharmacodynamic and Toxicity Assess-ment. 2.4.1. Tumor Growth Inhibition Studies. FemaleSprague−Dawley (SD) rats of 220−230 g and 4−5 weeks oldwere supplied by the central animal facility (CAF), NIPER,India before commencement of the study. All animal studyprotocols were duly approved by the Institutional AnimalEthics Committee (IAEC) of National Institute of Pharma-ceutical Education & Research (NIPER), India. The animalswere acclimatized at a temperature of 25 ± 2 °C and relativehumidity of 50−60%, under a natural light/dark cycle for oneweek before experiments. For tumor induction, DMBA in soyabean oil was administered orally to rats at 45 mg/kg dose at aweekly interval for three consecutive weeks.18,30 Measurablemammary tumors of average size 1200−1500 mm3 wereobserved in animals after 10 weeks of the last DMBA dosingand tumor bearing animals were separated.For pharmacodynamic assessment, animals were divided into

five groups, each group containing four animals. Free DOX,DOX, DOX@m-PEG-MWCNTs, and DOX@E2-PEG-MWCNTs (normalized to 5 mg/kg of free DOX) wereadministered to the first four groups of animals via intravenousinjection. The last group was kept as the control, whichreceived normal saline in a similar way. The tumor volume andbody weight was measured on every alternate day posttreatment by Vernier caliper using the following equation:

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tumor volume (V) = D*d2/2, where d is the smallest and D isthe longest length of the tumor. The study was terminated onthe 10th day after a single injection treatment. Blood wascollected through cardiac puncture for analyzing biochemicalmarkers of toxicity. Finally, animals were humanely sacrificed.Tumors were excised from the sacrificed animals and washedwith Ringer’s solution to remove any adherent debris and driedusing tissue paper. The photographs of excised tumors werecaptured and weighed using an electronic balance. Thereafter,excised tumors from various treatment groups were sliced intosmall pieces and subjected to high speed homogenization. Afraction of the resultant homogenate was diluted with PBS andsonicated for 2 min to aid in homogeneous dispersion ofsuspended particles. Finally, the optical density (OD) of thehomogenates was measured at 550 nm, and intratumoralconcentration of CNTs was determined from a standard plot,following the same protocol depicted in our earlier reports.Tumor homogenate of the control (untreated) group wassubtracted from the OD of the treated groups in order to nulifythe affect of other biomacromolecules (if any) absorbing at thesame wavelength.2.2.5.2. Toxicity Studies. In order to address the toxicity

issues pertaining to free DOX and functionalized MWCNTs,the various parameters for cardiotoxicity, hepatotoxicity, andnephrotoxicity were evaluated in tumor-induced rats. Prior tosacrifice of animals treated with free DOX and variousMWCNT preparations after 10 days’ treatment, blood sampleswere collected via cardiac puncture into heparinised capillarytubes. Plasma was separated by centrifugation at 10000g for 10min and stored at −20 °C until analysis. Enzyme activities suchas CK-MB and LDH levels were analyzed in plasma while SODwas determined in heart homogenate using commerciallyavailable kits based on the method provided by themanufacturer instructions supplied with the commercial kits.The various hepatotoxicity (AST, ALT, SOD, etc.) andnephrotoxicity (BUN) parameters were evaluated followingour earlier reported protocol.39

2.5. Statistical Analysis. All data unless otherwise specifiedare expressed as mean ± SD. Statistical analysis was performedwith Graph Pad Prism (version 6.03, USA) using one-wayANOVA followed by the Tukey−Kramer multiple comparisontest. P < 0.05 was considered as statistically significant.

3. RESULTS

3.1. Development and Characterization of E2-PEG-MWCNTs. In this study, functionalization of MWCNTs wasexecuted by covalent conjugation of carboxylated MWCNTswith a newly synthesized, amine-terminated E2-PEG derivative(Figure 1). FT-IR spectrum of E2-anchored MWCNTscompared to PEGylated E2 and E2-hemisuccinate have beenpresented as Figure S1 of the Supporting Information. In theFT-IR spectrum of E2-hemisuccinate, a sharp doublet wasdocumented at 1736 cm−1. These bands were assigned to thecarboxyl (γ CO) and ester (γ −O−CO) stretchingvibrations of the succinyl spacer, superimposed with oneanother. After PEGylation, a broad band was observed at 1758cm−1. This band was also accompanied with a medium intensityband at 1626 cm−1, attributed to the formation of amide linkagebetween PEG and E2-hemisuccinate. As expected, broad,intense bands featuring −C−O vibrations of the PEG unitappeared at around 1097 cm−1. The center of the OHstretching vibration too shifted from 3324 to 3444 cm−1

substantiating successful derivatization of E2-hemisuccinatewith PEG. In the FT-IR spectrum of E2-PEG-MWCNTs, abroad band superimposed with a number of medium intensitybands was observed in the range of 1700−1500 cm−1. Thesebands represented characteristic stretching of the various amideand ester linkages interlinking E2-hemisuccinate, PEG, andcarboxylated MWCNTs. The 1H NMR spectrum of E2-PEG-NH2 revealed the presence of characteristic proton signals ofthe steroid aromatic ring at δ 7.1−7.0, 6.5−6.4, and 6.4−6.3ppm. In addition to the characteristic peaks for steroid moiety,distinctive proton signals of the PEG (−O−CH2−CH2) unitwere observed over the range of 3.8−3.2 ppm, substantiating to

Figure 2. (A) Confocal laser scanning images of MCF 7 cells treated with E2-PEG-MWCNTs in the absence (left two panels) and presence (righttwo panels) of E2. For each incubation type, panels (a) and (b) represent RITC fluorescence and overlay of RITC and DAPI fluorescence,respectively. The white line represents a 50 μm scale bar. (B) Cell uptake profile of A549 and MCF 7 cells incubated with E2-PEG-MWCNTs in theabsence and in presence of E2.

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successful derivatization of E2 with PEG. The presence of PEGin the final conjugate was also confirmed by 13C NMR analysisin which the characteristic peak of the polyoxyethylene (−O−CH2−CH2) carbons were documented between δ 60−65 ppm.Final confirmation regarding the structure of this newlysynthesized E2-PEG came from mass spectral analysis in theMALDI mode. The MALDI-TOF spectrum of E2-PEG-NH2

(Figure S2 of the Supporting Information) exhibited a bell-shaped distribution for fragment ion peaks with the center ofthe bell at m/z 3823. This value was close to the theoreticallycalculated molecular ion peak of the conjugate. A closerinspection of the spectrum revealed that the fragment ion peaksbetween m/z 3412 and 4251 followed an arithmeticprogression with approximate mean difference of 44. Thisdifference corresponded to the molar mass of one −O−CH2−CH2 unit of PEG. Having authenticated the molecular structureof our newly synthesized E2-PEG derivative, we proceeded forfine-resolved structural characterization of the various organicmolecules immobilized on the surface of MWCNTs. Figure S3of the Supporting Information presents the HRMAS-NMRspectrum of the E2-PEG-MWCNT conjugate compared to

plain oxidized MWCNTs. The 1H NMR spectrum of oxidizedMWCNTs is almost flat and noisy, excepting for the solventpeak for DMSO at δ 2.49 ppm. In the case of E2-PEG-MWCNTs, characteristic doublets of the phenyl ring appearedat 7.2 and 6.6 ppm. The chemical shifts at 3.3−3.4 ppm wereassigned to the −O−CH2−CH2 units of the PEG chain. Thevarious signals ranging between 2.7 and 1.1 ppm wererepresentative of the various methylene and methane protonsof the steroid rings. The sharp singlet at 0.9 ppm was ascribedto 11-methyl (−CH3) protons of the steroid moiety.Representative scanning electron microscopy (SEM) imagesof E2-PEG-MWCNTs and oxidized MWCNTs compared totheir aggregated, pristine (p-) counterpart has been presentedas Figure S4A of the Supporting Information. As evident fromthese micrographs, the length of f-MWCNTs ranged between300 and 600 nm. The high-resolution TEM image of oxidizedMWCNTs has been presented in Figure S4B of the SupportingInformation. Although acid-oxidation led to almost 3−4-foldshortening of CNTs’ length, no significant detrimentation instructural integrity was observed.

Figure 3. Representative confocal images of (A) NR (B) Rh123-stained A549 cells incubated with E2-PEG-MWCNTs for 3 h. The white linerepresents the 50 μm scale bar. For micrographs (A) and (B) (a, b) represents the line display of AF647 and NR/Rh123 fluorescence, (c) representsthe line plot of AF647 and NR/Rh123 fluorescence, and (d) the scattered plot. (C) Representative confocal images of DAPI stained (I, II) A549cells and (III, IV) MCF 7 cells incubated with RITC-labeled (I, III) m-PEG-MWCNTs and (II, IV) E2-PEG-MWCNTs for 3 h. The panels (a), (b),and (c) represent RITC fluorescence, overlay of DAPI and RITC fluorescence and (c) scattered plot, respectively. The horizontal and vertical axes ofeach scatter plot represents the values of pixels in channel 2 (ch2) and channel 1(ch1), respectively.

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3.2. In Vitro Cellular Uptake, Subcellular Trans-location, and Intranuclear Drug Delivery. Cell uptakeand intracellular trafficking of E2-PEG-MWCNTs was evaluatedin MCF 7 and A549 cells expressing high levels of ER.Representative confocal images of MCF 7 cells incubated withE2-PEG-MWCNTs in the absence and presence of E2 for 3 hare shown in Figure 2A. In either case, nanotubes showedreckonable internalization by the target cells, albeit somechanges in the degree of internalization were observed. Whilepretreatment of cells with E2 could not stop CNTs fromentering the cells, a notable decrease in the extent of nanotubeinternalization was observed. To further understand thecontribution of E2-functionalization on the subcellular trans-location of CNTs, A549 cells were incubated with E2-PEG-MWCNTs, and their colocalization with various cell organelles,including lysosomes, mitochondria, and nucleus, was studiedusing confocal microscopy.37 For lysosome and mitochondrialcolocalization experiments, f-MWCNTs were covalently taggedwith a maleimide ester of AF-647 (indicated as grayfluorescence), whereas lysosomes and mitochondria werestained with NR (red fluorescence) and Rh123 (greenfluorescence), respectively. For nuclear colocalization analysis,E2-PEG-MWCNTs were labeled with rhodamine B isothiocya-nate (RITC, red fluorescence), while the nucleus was stainedwith 4′,6-diamidino-2-phenylindole (DAPI, blue fluorescence).As depicted in our earlier studies,18 colocalization in the entirefield of view was determined through scattered plot analysis,generated using Olympus Fluoview. The extent of colocaliza-tion between AF-647/RITC-labeled MWCNTs and anyorganelle-specific fluorescence dye was expressed in terms ofPearson’s correlation coefficient (r). A colocalization coefficientclose to or greater than 0.5 (r ≥ 0.5) was considered to be anindicator of good colocalization.30 As evident from the linedisplay of AF-647-labeled E2-PEG-MWCNTs and NR/Rh123-stained lysosome/mitochondria (Figure 3, panels A and B), E2-PEG-MWCNTs had nominal propensity to accumulate inlysosome (r = 0.197) or mitochondria (r = 0.299). Of note, theE2-PEG-MWCNT-conjugate showed significant localization inthe perinuclear and nuclear region (r > 0.6) in contrast to theER nontargeted m-PEG-MWCNTs (r < 0.3) that were mainlyrestricted to cellular cytoplasm (Figure 3C). Similar resultswere observed in MCF 7 cells too, wherein the E2-targetedconjugates showed significant nuclear compartmentalization (r> 0.5). Table 1 summarizes the Pearson’s colocalizationcoefficient of E2-PEG-MWCNTs with various cell organellesin the absence and presence of E2. While E2 pretreatment ofcells had little or practically no effect on the colocalization ofE2-PEG-MWCNTs with lysosome or mitochondria, a signifi-cant change with respect to nuclear colocalization was observed

(Figure S5 of the Supporting Information). In the case of E2pretreatment, nanotubes were mainly distributed in the cellularcytoplasm with little or practically no colocalization with thecell nuclei.As a direct test of intranuclear drug delivery by E2-PEG-

MWCNTs, DOX was loaded onto the side walls of E2-PEG-MWCNTs and E2-nontargeted m-PEG-MWCNTs, respec-tively, by exploiting the supramolecular π−π stackinginteractions between the drug and nanotubes. The drug-loadingcapacity of m-PEG-MWCNTs and E2-PEG-MWCNTs wasdetermined using our earlier reported method26,30 and issummarized in Table 2. Conjugation of PEG/E2-PEG with

MWCNTs hardly influenced the loading or release character-istics of drug-loaded CNTs, compared to plain, oxidizedMWCNTs or our recently reported HA-MWCNTs30 (FigureS6 of the Supporting Information). To investigate the cellularinternalization and intranuclear releases of DOX, A549 cellswere incubated with DOX@ E2-PEG-MWCNTs at 37 °C for 3h. Confocal microscopy was done to visualize the releasedDOX/DOX@ f-CNTs. Figure 4A presents the confocalfluorescence image of single A549 cells incubated withDOX@ E2-PEG-MWCNTs. The micrographs (a−c) representthe DOX fluorescence, overlay of DOX fluorescence, anddifferential interference contrast (DIC) image of DOX@E2-PEG-MWCNTs incubated cells. As evident from the images,the red fluorescence of DOX was highly accumulated in thenuclear and perinuclear region indicative of the presence ofDOX/DOX-nanotubes inside as well as in the vicinity of thecell nuclei. To further validate our hypothesis, nuclearcolocalization studies were also performed in a second ER(+ve) cell line (i.e., MCF 7) (Figure 4B). The nuclei of cellswere stained with DAPI while DOX@f-CNTs was detectableby their intrinsic red fluorescence. In line with our expectation,both free DOX and DOX@m-PEG-MWCNTs presented littleor practically no red fluorescence in the nuclei (Figure 4B,sections a and b). Contrastingly, DOX@E2-PEG-MWCNTsshowed intense red fluorescence in the nucleus (Figure 4B,section c), as evident by a high Pearson colocalizationcoefficient (r > 0.6) value. The intranuclear delivery of DOXvia E2-PEG-MWCNTs was further confirmed by alteration innuclear morphology as evidenced by condensed nuclei. Theappearance of condensed nuclei, an important hallmark of earlyapoptosis, was featured only in DOX@E2-PEG-MWCNT-treated cells (Figure 4C); no changes or abnormalities were

Table 1. Pearson’s Colocalization Coefficient of Various f-CNTs with Various Cell Organellesa

Pearson’s colocalization coefficient (r)

S(no.)

formulation(s)tested lysosome mitochondria nucleus

1 E2-PEG-MWCNT-DOX (ER−)

0.197 ± 0.016 0.299 ± 0.023 0.62 ± 0.08

2 E2-PEG-MWCNT-DOX (ER+)

0.186 ± 0.012 0.274 ± 0.026 0.27 ± 0.09

aData represents mean ± SEM (n = 3).

Table 2. Half Maximal Inhibitory Concentration (IC-50) ofFree Drug and Drug Loaded f-CNTsc

IC-50 (μg/mL)d

sampleexamined

entrapmentefficiency (EE

%)cdrug loading

(%)c A549 MCF 7

DOX (free) − − 2.4 ± 0.3a 2.3 ± 0.2a

m-PEG-MWCNT-DOX

98.9 ± 0.6 31.6 ± 0.2 1.6 ± 0.2b

a***1.5 ± 0.1b

a***

E2-PEG-MWCNT-DOX

99.3 ± 0.5 32.1 ± 0.1 1.0 ± 0.1a***,b***

0.9 ± 0.1a***,b***

cEE (%) and drug-loading data expressed as mean ± SD (n = 6).dData represents mean ± SEM of three experiments (n = 3, perconcentration per experiment).

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observed in cells treated with either nontargeted conjugate/freedrug (data not shown).3.3. Anticancer Activity of DOX@E2-PEG-MWCNTs: A

Correlation with Nuclear Targeting. To find any possibleenhancement of anticancer efficiency of DOX@E2-PEG-MWCNTs, both A549 and MCF 7 cells were incubated withfree DOX, DOX@PEG-MWCNTs, and DOX@E2-PEG-MWCNTs for 24 h. Cytotoxicity was evaluated by the MTTassay. The IC-50 values of free DOX and DOX@f-CNTs(normalized to DOX concentration) are summarized in Table2. The order of cytotoxicity, as determined from the assay, wasDOX@E2-PEG-MWCNTs > DOX@m-PEG-MWCNTs >DOX. It is, however, worthy to mention that DOX-deprivedformulations presented little or practically no toxicity, even atconcentrations greater than 100 μg/mL. To further validate theER targeting capability of the synthesized conjugate, cytotox-icity study was also performed by pre-exposing the cells toexcess (50 μg/mL) of E2, followed by incubation with DOX@E2-PEG-MWCNTs. In the presence of E2, the cytotoxicity ofthe conjugate was significantly depreciated, and the observedgrowth inhibition (%) was even less than 10. To furthervalidate the concept of nuclear targeting, A549 cells wereincubated with free DOX, DOX@PEG-MWCNTs, and DOX@E2-PEG-MWCNTs for 24 h, following which apoptosis andDNA fragmentation was quantified using TUNEL and DPAassays. As evident from Table 3, DOX@E2-PEG-MWCNTs

exhibited higher TUNEL (+ve) cells and fragmented DNA(75.2 ± 6.8%; 59.2 ± 5.5%) as compared to both nontargetedconjugate (56.2 ± 4.8%; 35.2 ± 3.1%) and free drug (52.3 ±5.2%; 31.3 ± 3.9%).

3.4. Pharmacodynamic and Toxicity Assessments.Finally, we tried to elucidate whether direct, intranucleardelivery of DOX through E2-PEG-MWCNTs could enhancethe therapeutic efficacy of DOX in vivo. Subsequently,pharmacodynamic studies were carried out in chemicallyinduced breast cancer using DMBA in female Sprague−Dawley(SD) rats. Animals were administered with 5 mg/kg of free

Figure 4. (A) Representative single-cell imaging of (A) A549 cells incubated with DOX@E2-PEG-MWCNTs. The images (a−c) represent the DOXfluorescence, overlay of DOX fluorescence, and differential interference contrast (DIC) image of cells and 3D reconstruction of a DOX@ E2-PEG-MWCNTs internalized cell. (B) Representative confocal micrograph of (A) MCF 7 cells incubated with (a) free DOX, (b) DOX@m-PEG-MWCNTs, and (c) DOX@E2-PEG-MWCNTs. The panels (i−iii) represent the DOX fluorescence, overlay of DOX fluorescence and DAPIfluorescence, and scattered plot of red and blue channels for the entire field of view. The horizontal and vertical axes of each scatter plot representsthe values of pixels in channel 2 (ch2) and channel 1 (ch1), respectively. (C) DOX@E2-PEG-MWCNTs induced apoptotic induction of MCF 7cells.

Table 3. Quantification of f-CNT Induced Apoptosis in A549Cellsd

apoptotic cells (%) fragmented DNA (%)

sample examined TUNEL assay DPA assay

control 5.2 ± 1.5a 1.98 ± 0.22a

DOX (free) 52.3 ± 5.2b a*** 31.3 ± 3.9 a***m-PEG-MWCNT-DOX

56.2 ± 4.8c a*** 35.2 ± 3.1 a***

E2-PEG-MWCNT-DOX

75.2 ± 6.8 a***, b*, c* 59.2 ± 5.5 a***, b*, c**

dData represents mean ± SEM (n = 3). a*, b*, and c* representsquantification of various apoptotic markers with respect to control,free DOX, and m-PEG-MWCNT-DOX, respectively (***p < 0.001,**p < 0.01, *p < 0.05).

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DOX, DOX@m-PEG-MWCNTs, and DOX@E2-PEG-MWCNTs (normalized to 5 mg/kg of free DOX). Figure 5A

presents the tumor growth inhibitory effect of free DOX/DOX@f-CNTs in SD rats. A single intravenous (i.v.)administration of DOX@E2-PEG-MWCNTs led to a rapiddecline in tumor burden within 24 h of administration. Overthe course of first 4 days’ treatment, both free DOX and DOX-loaded CNTs led to a steady decrease in tumor burden incontrast to saline or plain E2-PEG-MWCNTs treated groupshowing continuous increase in tumor growth. On the sixthday, the minimized tumor volumes of rats treated with freeDOX (87.36 ± 5.4%) differed significantly (p < 0.01) fromDOX@m-PEG-MWCNTs (52.43 ± 3.65%) and DOX@E2-PEG-MWCNTs (36.29 ± 3.45%). Thereafter, tumor volume ofthe free DOX-treated group started increasing while drug-loaded CNTs maintained a constant reduction in the tumor

volume. The minimized tumor volume in rats after 10 days oftreatment with DOX@E2-PEG-MWCNTs was approximately18, 17, 5, and 2 times less as compared to rats treated withsaline, drug-deprived E2-PEG-MWCNTs, free DOX, andDOX@m-PEG-MWCNTs, respectively. Representative photo-graphs illustrating the reduction of the tumor burden in DOX@E2-PEG-MWCNTs-treated rats have been presented as Figure5B. The study, however, was terminated after 10 days, as nosignificant changes in tumor volume was observed in between 6and 10 days. Consequently, animals were sacrificed on the 10thday and tumors were excised from sacrificed animals. Figure 5Cpresents the photographs of excised tumors treated with freeDOX/DOX-fMWCNTs. While control and free DOX-treatedtumor mass retained their original color, black carbon-basedparticles were uniformly distributed in the excised tumorspretreated with m-PEG-MWCNT-DOX and E2-PEG-MWCNT-DOX. The intratumoral concentration of CNTsper unit mass of tumor homogenate was determinedspectrophotometrically using an already standardized protocoldescribed in our laboratory. As obvious, no traces of CNTscould be detected in either control- or DOX-treated groups.Interestingly, around 56.32 ± 4.67 and 78.26 ± 6.83 μg ofCNTs per milligram of tumor homogenate were detected form-PEG-MWCNT-DOX and E2-PEG-MWCNT-DOX, whichcorresponded to approximately 21.56% and 32.45% of theinjected dose per unit mass of CNTs.A major limitation of DOX therapy is its propensity to

aggravate cardiotoxicity, following iv injection. We, therefore,sought to evaluate acute toxicity in rats exposed to both freeDOX and DOX-loaded CNT formulations for 15 days. Aftertermination of the pharmacodynamic study on the 10th day,the various cardiotoxicity markers, including lactate dehydro-genase (LDH), cytokinin MB (CK-MB), and aspartatetransaminase (AST) levels in plasma and superoxide dismutase(SOD) activity in heart homogenate were determined. Anincrease in LDH, CK-MB, or AST level and decreased SODactivity indicates cardiotoxicity. As evident from the variouscardiotoxicity marker levels, DOX delivered through CNTsshowed marked depreciation in cardiotoxicity in comparison tothe free drug. As apparent from Figure 6 (panels a−b),following 10 days of treatment, the LDH level in heart tissueand CK-MB level in the plasma were significantly increased inthe animal groups treated with free DOX. Conversely, LDHand CK-MB levels of various f-CNT-treated groups showedinsignificant differences from the control group. In line with theresults of other cardiotoxicity assessment parameters, AST levelof free DOX treated rats showed significant elevation, indicativeof myocardial infarction, whereas rats treated with f-CNTspresented insignificant differences from the control (Figure 6c).Concurrently, SOD levels in the heart homogenate of freeDOX-treated group decreased while both DOX@m-PEG-MWCNTs and DOX@E2-PEG-MWCNTs presented compa-rable SOD activity with respect to control (Figure 6d). In thisconnection, it may be noted that neither free DOX nor DOX-loaded CNTs did present any detectable hepatoxicity ornephrotoxicity over the course of treatment.

4. DISCUSSIONSIn this study, E2-PEG-MWCNTs were synthesized by covalentcoupling of amine-terminated E2-PEG with carboxyl-enrichedMWCNTs. While both single- and multiwalled carbonnanotubes have sparked interest in their use as therapeuticvectors, we sought to proceed with MWCNTs because the

Figure 5. (A) In vivo tumor growth inhibition profile of SD ratsintravenously administered with free DOX and various functionalizedMWCNT preparations loaded with an equivalent amount of the freedrug. (B) Representative photograph of tumor reduction in ratstreated with DOX@E2-PEG-MWCNTs over time. (C) Representativephotograph and spectrophotometric data showing the intratumoralpresence of DOX@f-MWCNTs.

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latter presents a wider surface that permits a more efficientinternal encapsulation and external functionalization with activemolecules as compared to their single-walled counterparts.40

Further, in a number of studies focusing on nanotoxicology ofCNTs, it has been found that MWCNTs are less toxic ascompared to SWCNTs, which make them an ideal choice for invivo applications.41,42 The successful immobilization of E2-PEGon the surface of carboxylated MWCNTs was substantiatedthrough FT-IR and HRMAS-NMR analysis. The intracellularuptake of E2-PEG-MWCNTs by ER(+ve) MCF 7 cells wasstudied using confocal microscopy. As expected, E2-PEG-MWCNTs presented appreciable internalization by itsmolecular target. To further apprehend the contribution ofERs in cellular internalization of E2-conjugated MWCNTs,competitive inhibition studies were performed. While E2-pretreatment could not restrain CNTs from entering the cells,marked changes with regards to intracellular concentration ofE2-PEG-MWCNTs was recorded. In this regard, it may benoted that although classical ERs are nucleocytoplasmic,43,44

some nonnuclear ER-αs are localized on the cell surfacemembrane as well. These membrane-bound ERs, combinedwith other energy-driven factors, altered the cellular uptake (%)and intracellular distribution of E2-PEG-MWCNTs.Our next step was to have a look at the intracellular

trafficking of E2-PEG-MWCNTs. For this purpose, we selectedA549 cells as our primary molecular target because this cell linepresents a flat morphology, which is favored for intracellularcolocalization analysis. Our results showed that E2-PEG-MWCNTs had nominal proclivity to accumulate in lysosomeor mitochondria but significant propensity toward nuclearcompartmentalization. These results strengthened our expect-ations regarding the active intervention of ERs in theintracellular uptake as well as the nuclear localization of drug-loaded nanotubes. It seemed that the presence of E2 on thesurface of CNTs facilitated the binding of the conjugate with

membrane-associated and cytoplasmic ERs, which in turn,assisted their energy-dependent transport across the nuclearmembrane (Figure 3C). Although the diameter of E2-PEG-MWCNTs (30−50 nm) was too large to permeate across thenuclear pore (10−20 nm), steroids have been reported to dilateor perforate the nuclear pore (between 90 and 120 nm)through formation of nuclear−pore complexes (NPC)45.46 Thetransport of hydrophobic cargoes such as ER-E2-PEG-MWCNTs (receptor−ligand) complex is believed to alter thepermeability and conformational state of the NPCs, therebyinitiating nuclear trafficking. At this point, it was necessary toexamine the merits of nuclear-localized E2-PEG-MWCNTs interms of their capability of intranuclear drug delivery andrelease. To have a proof-of-concept, DOX was loaded onto thesidewalls of m-PEG-MWCNTs and E2-PEG-MWCNTs,following which their cellular internalization and intranuclearrelease was investigated in two different ER (+ve) cell lines:A549 and MCF7. In case of DOX, very slight red fluorescencewas visible either in the cell cytoplasm or the nucleus (Figure4B, section a). Such observation is not against streamlinebecause the overexpression of p-glycoprotein (Pgp) receptorson MCF 7 cells47 may limit the internalization of the free druginside the cells. For m-PWG-MWCNT-DOX, red fluorescencewas spread throughout the cell cytoplasm with very little redfluorescence in nuclei due to a concentration gradient diffusionmechanism of free DOX. Contrastingly, intense red fluo-rescence was visible in the nuclei of cells incubated withDOX@E2-PEG-MWCNTs. We inferred that free DOX or E2-deprived DOX@m-PEG-MWCNTs cannot be directly deliv-ered into the nuclei in the absence of any energy-dependent,receptor-mediated pathway. Likewise, DOX released from m-PEG-MWCNTs in the cytoplasm is slowly diffused into thenucleus to exert therapeutic activity. However, as already shownin Figures 2 and 3, E2-PEG-MWCNT-conjugate presented theunique capability to localize in the cell nucleus, which enabled

Figure 6. Evaluation of various cardiotoxicity ad hepatotoxicity parameters: (a) CK-MB, (b) LDH, (c) AST/ALT, and (d) % SOD in tumor-bearingrats treated with free DOX/DOX-loaded MWCNTs [a* with respect to control (***p < 0.001, **p < 0.01, *p < 0.05)].

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the conjugate to directly release the loaded drug at the nucleusitself. It can also be known that the red fluorescence in Figure4B (section c) is from DOX@E2-PEG-MWCNT-conjugaterather than that of free DOX that had already been released inthe cytosol and then diffused into nuclei. The aboveobservations are more qualitatively confirmed by the scatteredplot of fluorescent intensity of selected MCF7 cells, as shown inFigure 4B. The intranuclear localization of DOX@E2-PEG-MWCNTs ensures slow, sustained release of DOX specificallyto its pharmacodynamic site of action so that hallmarks ofapoptosis (viz. condensed nuclei) were visible as early as 3 h(Figure 4C). In attempts to find the possible enhancements inanticancer efficacy by E2-PEG-MWCNTs, we observed thatDOX-loaded MWCNTs, in general, exerted higher cytotoxicitycompared to free DOX (Table 1). The higher cytotoxicity ofDOX@mPEG-CNTs compared to free DOX may beaccounted to higher intracellular availability of DOX, deliveredthrough an intrinsically cell-penetrable carrier like CNTs.Between plain DOX@m-PEG-MWCNTs and DOX@E2-PEG-MWCNTs, the IC-50 of the ER-targeted conjugate in A549 andMCF 7 cells was 1.5−1.6 times lower than its nontargetedcounterpart (P < 0.001) which, however, seems to be aconsequence of direct, intranuclear delivery of DOX intervenedby E2-PEG-MWCNTs that was not operative in the case of thenontargeted conjugate. Depreciation in cytotoxicity in the caseof E2 pretreatment revalidates that the presence of E2 on thesurface of MWCNTs play a key role in targeting DOX to its siteof action (i.e., nucleus). This hypothesis was revalidated fromapoptosis studies in which DOX@m-PEG-MWCNTs showedhigher TUNEL(+ve) cells (a hallmark of late apoptosis) andfragmented DNA compared to both free drug and nontargetedcontrol.Having established the merits of our newly synthesized

carrier system with regards to cell-selective, intranuclear drugdelivery, we tried to understand whether the effect can betranslated into an equivalent therapeutic response in vivo. Inline with that approach, pharmacodynamic studies wereconducted in chemically, breast tumor-induced rats. DMBAwas used as the carcinogen. We opted to work on this particularmodel because DMBA is widely used to induce breast tumorsin rodents with the average rate of tumor induction varyingfrom 65 to 75%. Finally, there have been reported evidence ofestrogen receptor overexpression in DMBA-derived mammarytumors in female SD rats.48 In this regard, it is worth notingthat among all the treatment groups, rats treated with DOX@E2-PEG-MWCNTs exhibited the highest antitumor activity,which is in line with the results of in vitro cytotoxicityexperiments. In addition, intratumoral presence of CNTs at thetumor site was confirmed through spectrophotometric analysisof f-CNTs in the tumor homogenate, which was furthersupported through visual observations. The higher intratumoralconcentration of DOX@E2-PEG-MWCNTs over DOX@m-PEG-MWCNTs may be attributed to its prolonged intra-tumoral presence, facilitated through ligand−receptor bindinginteraction. As ERs are overexpressed in more than 80% ofbreast tumors, the estradiol moiety present on the surface ofMWCNTs can potentially interact and bind with cytoplasmicERs. This interaction protracts the intracellular retention ofdrug-loaded nanotubes. The higher antitumor activity ofDOX@E2-PEG-MWCNTs compared to DOX@m-PEG-MWCNTs or free DOX may be attributed to their highintratumoral accumulation and prolonged intracellular avail-ability, facilitated by (i) enhanced permeability and retention

(EPR) effect of the hydrophilic PEG spacer interconnecting E2and MWCNTs (passive targeting), (ii) intrinsic cell pene-trability of MWCNTs via the well-established “nanoneedlepathway”, and (iii) the slow, sustained intranuclear drugdelivery via an ER-mediated pathway. Direct transport of thechemotherapeutic agent to nucleus is not possible in the case ofE2-deprived PEGylated MWCNTs so that antitumor efficacy ofthe conjugate is somewhat compromised and is twice less thanits targeted counterpart. These results suggest that combinationof MWCNTs, PEG, E2, and DOX into a single system results ina synergistic anticancer response, justifying the rationale of ourcarrier design. While free DOX treatment induced severecardiotoxicity in animals, animals treated with DOX@m-PEG-MWCNTs and DOX@E2-PEG-MWCNTs were devoid of anydetectable hepatotoxicity, nephrotoxicity, and cardiotoxicity.Cumulatively, these results suggest that the delivery property ofMWCNTs was effectively combined with ER avidity of E2 tofacilitate intranuclear drug delivery to breast tumors, whilemitigating drug as well as carrier-associated toxicity.

5. CONCLUSIONIn conclusion, we have prepared a novel, E2-PEG-MWCNTconjugate that can selectively target ER overexpressing cancercells and facilitate direct, intranuclear drug delivery through anER-dependent pathway. By loading DOX onto the sidewalls ofE2-PEG-MWCNTs, we demonstrated that conjugation of E2with MWCNTs not only augmented the nuclear targetingindex of carrier-bound drug but also synergized its therapeuticefficacy both in vitro and in vivo while alleviating drug-associated cardiotoxicity. The therapeutic conjugate was furtherintegrated with an organic fluorophore viz. AF-647/RITC toenable real-time monitoring of intracellular, organelle-specificlocalization of the targeted nanoprobe through opticalfluorescence imaging. For the first time, we have embarkedon the feasibility of cancer cell-selective, nuclear-targeted drugdelivery using steroid-conjugated MWCNTs as the moleculartransporter.Although further studies are required to elucidate the

detailed mechanism of nuclear penetration and long-term fateof E2-PEG-MWCNTs in vivo, proof-of-concept realized fromthe present study can be extended to augment the second- andthird-order targeting of a myriad of antineoplastic drugs, whichexert their pharmacodynamic action on the nucleus.

■ ASSOCIATED CONTENT*S Supporting InformationFT-IR, MALDI-TOF and HRMAS-NMR mass spectra, SEMand TEM images, confocal images, and drug release profile.This material is available free of charge via the Internet athttp://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: sanyogjain@niper.ac.in, sanyogjain@rediffmail.com.Tel: +91172-2292055. Fax: +91172-2214692.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSFinancial support for this work was provided by Indian Councilof Medical Research (ICMR), Government of India (GOI),New Delhi (Grant 35/28/2010/-BMS). M.D. and R.P.S. are

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grateful to the Department of Science and Technology (DST),GOI, New Delhi for providing postdoctoral and senior researchfellowships, respectively. Director NIPER is acknowledged forproviding necessary infrastructural facilities. Thanks are due toDr. Ravi S. Amarpati, SAIF, Central Drug Research institute(CDRI, Lucknow) for assistance with HRMAS-NMR analysis.The technical assistance of Mr. D.S. Chauhan and Mr. R.R.Mahajan is highly appreciated.

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