Intranuclear Drug Delivery and Effective in Vivo Cancer Therapy via

Article pubs.acs.org/molecularpharmaceutics

Intranuclear Drug Delivery and Effective in Vivo Cancer Therapy via Estradiol−PEG-Appended Multiwalled Carbon Nanotubes Manasmita Das, Raman Preet Singh, Satyajit R. Datir, and Sanyog Jain* Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar (Mohali), Punjab 160062, India S Supporting Information *

ABSTRACT: Cancer cell-selective, nuclear targeting is expected to enhance the therapeutic efficacy of a myriad of antineoplastic drugs, particularly those whose pharmacodynamic site of action is the nucleus. In this study, a steroidmacromolecular bioconjugate based on PEG-linked 17βEstradiol (E2) was appended to intrinsically cell-penetrable multiwalled carbon nanotubes (MWCNTs) for intranuclear drug delivery and effective breast cancer treatment, both in vitro and in vivo. Taking Doxorubicin (DOX) as a model anticancer agent, we tried to elucidate how E2 appendage influences the cell internalization, intracellular trafficking, and antitumor efficacy of the supramolecularly complexed drug. We observed that the combination of DOX with E2-PEGMWCNTs not only facilitated nuclear targeting through an estrogen receptor (ER)-mediated pathway but also deciphered to a synergistic anticancer response in vivo. The antitumor efficacy of DOX@E2-PEG-MWCNTs in chemically breast cancer-induced female rats was approximately 18, 17, 5, and 2 times higher compared to the groups exposed to saline, drug-deprived E2-PEGMWCNTs, free DOX, and DOX@m-PEG-MWCNTs, respectively. While free DOX treatment induced severe cardiotoxicity in animals, animals treated with DOX@m-PEG-MWCNTs and DOX@E2-PEG-MWCNTs were devoid of any perceivable cardiotoxicity, hepatotoxicity, and nephrotoxicity. To the best of our knowledge, this is the first instance in which cancer cellselective, intranuclear drug delivery, and, subsequently, effective in vivo breast cancer therapy has been achieved using estrogenappended 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 been identified as one of the most vital hormones regulating the development and maintenance of the female reproductive system and secondary sex characteristics.1−3 Binding of E2 with estrogen receptors (ER) induces conformational changes and release of molecular chaperone (Hsp 90, Hsp 70, cyclophilin, and p23) from the receptors,4 which allows them to conscript the cofactors necessary for transcription of various genes commonly upregulated in malignant cells (e.g., transforming growth factor alpha, c-myc, or cathepsin D). As evident from an extensive literature survey, hormone receptors like ERs and progesterone receptors are overexpressed in 70−80% of all breast cancers. The upregulation of ERs in cancerous cells relative to normal cells can be effectively harnessed for the development of a targeted therapy against various hormone sensitive cancers.5,6 ER-α is known to localize in both nucleus as well as plasma membrane, mediating estrogen-dependent, genomic, and nongenomic signaling.4,7−10 Subsequently, conjugation of estrogen hormones with any pharmaceutically active component (either free or carrier bound) may simultaneously facilitate cellular and intracellular, organelle© XXXX American Chemical Society

specific (nuclear) targeting in such receptor overexpressed cancer cells. A large number of first line chemotherapeutic medications, including doxorubicin (DOX) and cisplatin (CDDP), exert their pharmacodynamic effects in nuclei by intercalating with DNA base pairs, thereby inhibiting cell growth/proliferation.11 Unfortunately, the transport of these anticancer drugs from plasma membrane to nucleus is not very well-characterized. In fact, transport of drugs to nuclei have been found to be rather difficult, and even if it could happen, it is considered to be nonspecific and passive. Additionally, drug-resistant cancer cells have many intracellular drug-resistance mechanisms that avert the access of anticancer agents to the nucleus. Consequently, only a small percentage of the administered dose can be delivered into the cytosol and finally reach the nucleus.9 Over the past years, a number of ER-targeted bioconjugates have been prepared by coupling estrogens with a myriad of cytotoxic Received: April 21, 2013 Revised: July 7, 2013 Accepted: August 1, 2013

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dx.doi.org/10.1021/mp4002409 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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

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 largely compromised due in part to the modification of the parent estradiol molecule and addition of appendages of varying chemical nature and steric bulk. Subsequently, the desired degree of accumulation of the conjugate selectively into the tumor often remains unachieved. On the other side, a myriad of nano drug delivery systems have been developed to target cancer cells, but only limited recent reports have focused on the feasibility of nuclear targeting using nanoparticles.17,18 In those few instances too, the investigations were restricted to mere in vitro evaluations so that it is not possible to predict whether direct intranuclear release of therapeutic drugs in vitro could be deciphered into a comparable therapeutic response in vivo. At the same time, a few recent reports have embarked on the

feasibility of targeted cancer therapy using steroids anchored liposomes/gold nanoparticles as the therapeutic vectors.19−21 However, from the reported studies, it is not clear whether combination of an estrogen/estrogen antagonist with the nanocarrier could deliver the drug directly into the nucleus. We reasoned that tumor-specific accumulation as well as intranuclear drug delivery could be significantly enhanced, if the drug molecule of interest is loaded into an E2-anchored nanocarrier that has a natural propensity for intracellular penetration in addition to energy-dependent internalization via ligand−receptor affinity interactions. Amidst the innumerable nanocarriers that have elicited promise with regard to targeted drug delivery or imaging, functionalized carbon nanotubes (fCNTs) have sparkled phenomenal interest.22−25 The unique physicochemical and structural properties of these nanocarriers enable multiple diagnostic and therapeutic moieties to be B



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(NCSS), Pune, India. Maleimide ester of Alexa-Fluor 647 (AF647) was purchased from Invitrogen. All other chemicals/ solvents were of analytical grade and procured from local suppliers unless otherwise stated. 2.2. Methods. 2.2.1. Synthesis and Structural Elucidation of E2-PEG-MWCNT-Conjugate. The synthesis of E2-PEGMWCNT-conjugate was accomplished in three steps as depicted in Figure 1. Chemical structures of all synthesized compounds including final product and intermediates were preliminarily analyzed by Fourier transform-infrared (FT-IR) spectroscopy and further authenticated via proton (1H) and carbon (13C) nuclear magnetic resonance (NMR) spectroscopy. The molecular mass of the E2-PEG derivative was determined via matrix-assisted laser desorption ionizationtime-of-flight (MALDI-TOF) mass spectroscopy. The presence of various functional groups on the surface of MWCNTs was preliminarily studied through FT-IR, while fine-resolved structural characterization of surface-bound ligands was done through high-resolution magic angle spinning (HRMAS) NMR spectroscopy. Samples for HRMAS NMR experiments were prepared by suspending 10 mg of each nanoparticle preparation 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 mm HRMAS probe. 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 reported by Yellin but with minor modifications.29 Briefly, 17β-estradiol (300 mg, 1.1 mmol) was dissolved in anhydrous benzene (10 mL) and refluxed with approximately 5-fold molar excess of succinic anhydride (550 mg, 5.5 mmol) in the presence of pyridine (2 mL). The reflux was continued for approximately 24 h, when E2 was no longer detectable by thin layer chromatography (TLC). Upon cooling to room temperature, excess succinic anhydride precipitated out from the solution, which was filtered off. The filtrate was concentrated under reduced pressure by rotary evaporation, following which, the residue (E2-3, 17 disuccinate) was dissolved in methanol (10 mL) and stirred overnight with an excess of sodium bicarbonate (1 g suspended in 10 mL of water) to complete selective hydrolysis of the phenolic ester. Completeness of the reaction was 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 remove unreacted NaHCO3. After filtration of NaHCO3, water (10 mL) was added to the reaction mixture. The alkaline solution was extracted 3 times with diethyl ether (10 mL × 3). The aqueous phase was brought to pH 7 with 1 N HCl and then poured into a mixture of 0.1 N HCl and crushed ice. The white crystalline product that separated was removed by vacuum filtration, washed repeatedly with water, and air dried. The crude 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

integrated on the same nanotube platform, while their natural shape facilitates their noninvasive penetration across biological barriers. In one of our recently published reports, we have embarked on the feasibility of modulating the intracellular trafficking and therapeutic performance of drug-loaded carbon nanotubes by intelligent manipulation of their surface chemistry.26 In this study itself, we championed the idea of nuclear-specific drug targeting using estradiol-appended multiwalled carbon nanotubes (MWCNTs) as the therapeutic vector. However, our previous study was just a proof-ofconcept; we did not provide direct evidence of intranuclear drug release, and also our investigation was limited to mere in vitro evaluations. With motivation from our previous work, we sought to design a novel, nuclear-targeted, CNT-based bioconjugate that can potentially augment the therapeutic efficacy of its associated cytotoxin in vivo while minimizing drug-associated toxicities. In line with this idea, a novel, poly(ethylene glycol)-linked E2 derivative viz. 17β-estradiolhemisuccinyl-poly(ethylene glycol)-amine (E2-PEG) was synthesized and covalently conjugated to carboxylated MWCNTs. As established in earlier studies from our group as well as others’, we appended E2 and CNTs to the distal ends of a PEG spacer because such a surface design effectively (i) minimizes nonspecific sequestration of CNTs by the mononuclear phagocytic system (viz. liver and spleen) and (ii) increases carrier-localization to target tumor site via enhanced permeation and retention (EPR) effect (passive targeting).27,28 Taking DOX as a model anticancer agent, we tried to elucidate how E2 modification influences the cancer targetability, intracellular trafficking, and anticancer efficacy of the conjugate both in vitro and in vivo. We observed that combination of DOX with E2-PEG-MWCNTs not only facilitated intranuclear drug delivery through an ER-mediated pathway but could be effectively translated into a synergistic therapeutic response in vivo while reducing drug-associated cardiotoxicity. To our knowledge, this is the first example in which MWCNTs have been covalently tethered with a PEGylated E2 derivative and successfully explored for cancer cell selective intranuclear drug delivery, imaging, and effective therapy against breast cancer in vivo.

2. MATERIALS AND METHODS 2.1. Materials. Pristine (p) MWCNTs (purity >95%, length 1−5 μm, and diameter 20−30 nm) were procured from Nanovatech Pvt. Ltd., U.S. sulphuric acid, nitric acid (69− 72%), disodium hydrogen phosphate, sodium acetate, sodium bicarbonate, thionyl chloride, sodium lauryl sulfate, copper sulfate, and thiobarbituric acid were purchased from Loba Chemie Pvt. Ltd., India. Doxorubicin was obtained as a gift sample from Sun Pharmaceuticals, India. PEG bisamine (Mw = 3500) and methoxy-PEG were procured from JenKem Technology. 17β-Estradiol (E2), succinic anhydride, dicyclohexyl carbodiimide (DCC), N-hydroxysuccinimide (NHS), neutral red (NR), rhodamine 123 (Rh123), 4′,6-diamidino-2phenylindole dihydrochloride (DAPI), rhodamine B isothiocyanate (RITC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), and 7,12-dimethylbenz [α]anthracene (DMBA ≥ 95% pure) were purchased from Sigma. All kits for biochemical estimations were procured from Accurex, Biomedical Ltd., Mumbai. Culture medium and serum were procured 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 C



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PEG 3500 (in a 1.1 mol equiv of surface carboxyl density) for 24 h to afford the PEGylated conjugate in the desired yield. Yield: 75% (w/w). 2.2.2. Size and Morphology of MWCNTs. Microstructures of f-MWCNTs were analyzed using scanning electron microscopy (SEM, model S30400) and transmission electron microscopy (TEM, model FEI Tecnai G2). 2.2.3. DOX Loading and Release from MWCNTs. DOX loading on MWCNTs was consummated at pH 7.4 using the same protocol described in our earlier reports.30 Briefly, E2PEG-MWCNT and DOX were mixed in the ratio of 2:1 (w/w), and the resultant mixture was incubated at pH 7.4. The MWCNT−drug mixture was kept in a shaker bath at 37 °C for 24 h, following which, drug-loaded MWCNTs were separated by centrifugation and absorbance of the supernatant was measured at 480 nm. 2.3. In Vitro Cellular Uptake and Cytotoxicity Studies. Cell uptake studies were conducted in ER(+ve), A549, and MCF 7 cells.35 Briefly, cultured cells were exposed to various fMWCNT-formulations (10 μg/mL) for 1 h. Cell internalization of MWCNTs was visualized via confocal microscopy (Olympus FV 1000 microscope) and quantified using standard spectrofluorimetry-based techniques. In order to apprehend the roles of ER with regards to intracellular uptake and subcellular translocation of the synthesized conjugate, A549 cells were exposed to 50 μg/mL of different nanotube preparations in the absence and presence of E2 (50 μg/mL).36 Cell internalization was monitored via confocal microscopy. Intracellular colocalization studies were performed by labeling lysosomes, mitochondria, and nuclei of A549/HeLa/MCF 7 cells with neutral red (NR), Rh123, and DAPI, respectively, as detailed in our earlier reports.37,38 In vitro cellular cytotoxicity was preliminarily assessed by the conventional MTT assay. The IC-50 values were determined using Graph Pad Prism software (version 6.03). Free DOX and DOX-CNT-induced cellular apoptosis/DNA fragmentation were quantified using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and diphenylamine (DPA) assays. 2.4. In Vivo Pharmacodynamic and Toxicity Assessment. 2.4.1. Tumor Growth Inhibition Studies. Female Sprague−Dawley (SD) rats of 220−230 g and 4−5 weeks old were supplied by the central animal facility (CAF), NIPER, India before commencement of the study. All animal study protocols were duly approved by the Institutional Animal Ethics Committee (IAEC) of National Institute of Pharmaceutical Education & Research (NIPER), India. The animals were acclimatized at a temperature of 25 ± 2 °C and relative humidity of 50−60%, under a natural light/dark cycle for one week before experiments. For tumor induction, DMBA in soya bean oil was administered orally to rats at 45 mg/kg dose at a weekly interval for three consecutive weeks.18,30 Measurable mammary tumors of average size 1200−1500 mm3 were observed in animals after 10 weeks of the last DMBA dosing and 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@E 2 -PEGMWCNTs (normalized to 5 mg/kg of free DOX) were administered to the first four groups of animals via intravenous injection. The last group was kept as the control, which received normal saline in a similar way. The tumor volume and body weight was measured on every alternate day post treatment by Vernier caliper using the following equation:

(m, E2C9H, 1H), 0.7−0.6 (s, E2C18H, 3H). Mass: 372 (M + H). 2.2.1.2. PEGylation of E2-Hemisuccinate: Synthesis of E2PEG Derivative. The E2-hemisuccinate (42 mg, 0.1 mmol) in dichlormethane (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 PEGbisamine (350 mg/0.1 mmol dissolved in 1 mL of DCM) was added. After 24 h, a finely suspended, white precipitate of dicyclohyxyl urea (DCU) was envisaged in the reaction mixture, ensuring successful transformation of E2-hemisuccinate to the corresponding NHS ester. The precipitate was removed by filtration, following which the filtrate was added dropwise to ice cold ether (30−40 mL) to precipitate the PEGylated E2 derivative as a white semisolid. The crude product was air-dried, washed repeatedly with cold methanol to remove unreacted PEG-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); 1H NMR (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 E2Hemisuccinate. Carboxyl-enriched oxidized MWCNTs were prepared in accordance with our previous reports.30−32 For functionalization of MWCNTs with E 2 , acid-oxidized MWCNTs were converted to their corresponding acid-halide derivative by refluxing with thionyl chloride. Briefly, oxidized MWCNTs (100 mg) were dispersed in THF (10 mL) via ultrasonication for 1 min. To the resultant dispersion, SOCl2 (15−20 mL) was added, and the mixture was refluxed at 80 °C for 24 h.33 Thereafter, solvents were removed using rotavapor and the resultant acylated MWCNTs. As determined from thermo-gravimetric analysis (TGA), carboxylic density on the surface of MWCNTs was determined to be 0.0018 mmol/mg of MWCNTs. This value was necessary to calculate the exact amount of E2-PEG-NH2 that would be ideally required for complete interchange of surface carboxyl groups with the PEGylated steroid. On the basis of TGA results, around 3-fold molar 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. The reaction mixture was left stirring for 24 h, following which the nanotubes were isolated by centrifugation of the supernatant. The pellet of functionalized MWCNTs was purified by repeated washing with distilled water and acetone. Finally, the pellet was freeze-dried using an optimized freeze-drying cycle, recently patented by our group.34 Lyophilized MWCNTs were 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 mD



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Figure 2. (A) Confocal laser scanning images of MCF 7 cells treated with E2-PEG-MWCNTs in the absence (left two panels) and presence (right two 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 the absence and in presence of E2.

tumor volume (V) = D*d2/2, where d is the smallest and D is the longest length of the tumor. The study was terminated on the 10th day after a single injection treatment. Blood was collected through cardiac puncture for analyzing biochemical markers of toxicity. Finally, animals were humanely sacrificed. Tumors were excised from the sacrificed animals and washed with Ringer’s solution to remove any adherent debris and dried using tissue paper. The photographs of excised tumors were captured and weighed using an electronic balance. Thereafter, excised tumors from various treatment groups were sliced into small pieces and subjected to high speed homogenization. A fraction of the resultant homogenate was diluted with PBS and sonicated for 2 min to aid in homogeneous dispersion of suspended particles. Finally, the optical density (OD) of the homogenates was measured at 550 nm, and intratumoral concentration of CNTs was determined from a standard plot, following the same protocol depicted in our earlier reports. Tumor homogenate of the control (untreated) group was subtracted from the OD of the treated groups in order to nulify the affect of other biomacromolecules (if any) absorbing at the same 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, and nephrotoxicity were evaluated in tumor-induced rats. Prior to sacrifice of animals treated with free DOX and various MWCNT preparations after 10 days’ treatment, blood samples were collected via cardiac puncture into heparinised capillary tubes. Plasma was separated by centrifugation at 10000g for 10 min and stored at −20 °C until analysis. Enzyme activities such as CK-MB and LDH levels were analyzed in plasma while SOD was determined in heart homogenate using commercially available kits based on the method provided by the manufacturer instructions supplied with the commercial kits. The various hepatotoxicity (AST, ALT, SOD, etc.) and nephrotoxicity (BUN) parameters were evaluated following our earlier reported protocol.39

2.5. Statistical Analysis. All data unless otherwise specified are expressed as mean ± SD. Statistical analysis was performed with Graph Pad Prism (version 6.03, USA) using one-way ANOVA followed by the Tukey−Kramer multiple comparison test. P < 0.05 was considered as statistically significant.

3. RESULTS 3.1. Development and Characterization of E2-PEGMWCNTs. In this study, functionalization of MWCNTs was executed by covalent conjugation of carboxylated MWCNTs with a newly synthesized, amine-terminated E2-PEG derivative (Figure 1). FT-IR spectrum of E2-anchored MWCNTs compared to PEGylated E2 and E2-hemisuccinate have been presented as Figure S1 of the Supporting Information. In the FT-IR spectrum of E2-hemisuccinate, a sharp doublet was documented at 1736 cm−1. These bands were assigned to the carboxyl (γ CO) and ester (γ −O−CO) stretching vibrations of the succinyl spacer, superimposed with one another. After PEGylation, a broad band was observed at 1758 cm−1. This band was also accompanied with a medium intensity band at 1626 cm−1, attributed to the formation of amide linkage between PEG and E2-hemisuccinate. As expected, broad, intense bands featuring −C−O vibrations of the PEG unit appeared at around 1097 cm−1. The center of the OH stretching vibration too shifted from 3324 to 3444 cm−1 substantiating successful derivatization of E2-hemisuccinate with PEG. In the FT-IR spectrum of E2-PEG-MWCNTs, a broad band superimposed with a number of medium intensity bands was observed in the range of 1700−1500 cm−1. These bands represented characteristic stretching of the various amide and ester linkages interlinking E2-hemisuccinate, PEG, and carboxylated MWCNTs. The 1H NMR spectrum of E2-PEGNH2 revealed the presence of characteristic proton signals of the steroid aromatic ring at δ 7.1−7.0, 6.5−6.4, and 6.4−6.3 ppm. In addition to the characteristic peaks for steroid moiety, distinctive proton signals of the PEG (−O−CH2−CH2) unit were observed over the range of 3.8−3.2 ppm, substantiating to E



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Figure 3. Representative confocal images of (A) NR (B) Rh123-stained A549 cells incubated with E2-PEG-MWCNTs for 3 h. The white line represents the 50 μm scale bar. For micrographs (A) and (B) (a, b) represents the line display of AF647 and NR/Rh123 fluorescence, (c) represents the line plot of AF647 and NR/Rh123 fluorescence, and (d) the scattered plot. (C) Representative confocal images of DAPI stained (I, II) A549 cells 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 of each scatter plot represents the values of pixels in channel 2 (ch2) and channel 1(ch1), respectively.

plain oxidized MWCNTs. The 1H NMR spectrum of oxidized MWCNTs is almost flat and noisy, excepting for the solvent peak for DMSO at δ 2.49 ppm. In the case of E2-PEGMWCNTs, characteristic doublets of the phenyl ring appeared at 7.2 and 6.6 ppm. The chemical shifts at 3.3−3.4 ppm were assigned to the −O−CH2−CH2 units of the PEG chain. The various signals ranging between 2.7 and 1.1 ppm were representative of the various methylene and methane protons of the steroid rings. The sharp singlet at 0.9 ppm was ascribed to 11-methyl (−CH3) protons of the steroid moiety. Representative scanning electron microscopy (SEM) images of E2-PEG-MWCNTs and oxidized MWCNTs compared to their aggregated, pristine (p-) counterpart has been presented as Figure S4A of the Supporting Information. As evident from these micrographs, the length of f-MWCNTs ranged between 300 and 600 nm. The high-resolution TEM image of oxidized MWCNTs has been presented in Figure S4B of the Supporting Information. Although acid-oxidation led to almost 3−4-fold shortening of CNTs’ length, no significant detrimentation in structural integrity was observed.

successful derivatization of E2 with PEG. The presence of PEG in the final conjugate was also confirmed by 13C NMR analysis in 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 newly synthesized E2-PEG came from mass spectral analysis in the MALDI mode. The MALDI-TOF spectrum of E2-PEG-NH2 (Figure S2 of the Supporting Information) exhibited a bellshaped distribution for fragment ion peaks with the center of the bell at m/z 3823. This value was close to the theoretically calculated molecular ion peak of the conjugate. A closer inspection of the spectrum revealed that the fragment ion peaks between m/z 3412 and 4251 followed an arithmetic progression with approximate mean difference of 44. This difference corresponded to the molar mass of one −O−CH2− CH2 unit of PEG. Having authenticated the molecular structure of our newly synthesized E2-PEG derivative, we proceeded for fine-resolved structural characterization of the various organic molecules immobilized on the surface of MWCNTs. Figure S3 of the Supporting Information presents the HRMAS-NMR spectrum of the E2-PEG-MWCNT conjugate compared to F



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3.2. In Vitro Cellular Uptake, Subcellular Translocation, and Intranuclear Drug Delivery. Cell uptake and intracellular trafficking of E2-PEG-MWCNTs was evaluated in MCF 7 and A549 cells expressing high levels of ER. Representative confocal images of MCF 7 cells incubated with E2-PEG-MWCNTs in the absence and presence of E2 for 3 h are shown in Figure 2A. In either case, nanotubes showed reckonable internalization by the target cells, albeit some changes in the degree of internalization were observed. While pretreatment of cells with E2 could not stop CNTs from entering the cells, a notable decrease in the extent of nanotube internalization was observed. To further understand the contribution of E2-functionalization on the subcellular translocation of CNTs, A549 cells were incubated with E2-PEGMWCNTs, and their colocalization with various cell organelles, including lysosomes, mitochondria, and nucleus, was studied using confocal microscopy.37 For lysosome and mitochondrial colocalization experiments, f-MWCNTs were covalently tagged with a maleimide ester of AF-647 (indicated as gray fluorescence), whereas lysosomes and mitochondria were stained with NR (red fluorescence) and Rh123 (green fluorescence), respectively. For nuclear colocalization analysis, E2-PEG-MWCNTs were labeled with rhodamine B isothiocyanate (RITC, red fluorescence), while the nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI, blue fluorescence). As depicted in our earlier studies,18 colocalization in the entire field of view was determined through scattered plot analysis, generated using Olympus Fluoview. The extent of colocalization between AF-647/RITC-labeled MWCNTs and any organelle-specific fluorescence dye was expressed in terms of Pearson’s correlation coefficient (r). A colocalization coefficient close to or greater than 0.5 (r ≥ 0.5) was considered to be an indicator of good colocalization.30 As evident from the line display of AF-647-labeled E2-PEG-MWCNTs and NR/Rh123stained lysosome/mitochondria (Figure 3, panels A and B), E2PEG-MWCNTs had nominal propensity to accumulate in lysosome (r = 0.197) or mitochondria (r = 0.299). Of note, the E2-PEG-MWCNT-conjugate showed significant localization in the perinuclear and nuclear region (r > 0.6) in contrast to the ER nontargeted m-PEG-MWCNTs (r < 0.3) that were mainly restricted to cellular cytoplasm (Figure 3C). Similar results were observed in MCF 7 cells too, wherein the E2-targeted conjugates showed significant nuclear compartmentalization (r > 0.5). Table 1 summarizes the Pearson’s colocalization coefficient of E2-PEG-MWCNTs with various cell organelles in the absence and presence of E2. While E2 pretreatment of cells had little or practically no effect on the colocalization of E2-PEG-MWCNTs with lysosome or mitochondria, a significant change with respect to nuclear colocalization was observed

(Figure S5 of the Supporting Information). In the case of E2 pretreatment, nanotubes were mainly distributed in the cellular cytoplasm with little or practically no colocalization with the cell nuclei. As a direct test of intranuclear drug delivery by E2-PEGMWCNTs, DOX was loaded onto the side walls of E2-PEGMWCNTs and E2-nontargeted m-PEG-MWCNTs, respectively, by exploiting the supramolecular π−π stacking interactions between the drug and nanotubes. The drug-loading capacity of m-PEG-MWCNTs and E2-PEG-MWCNTs was determined using our earlier reported method26,30 and is summarized in Table 2. Conjugation of PEG/E2-PEG with Table 2. Half Maximal Inhibitory Concentration (IC-50) of Free Drug and Drug Loaded f-CNTsc IC-50 (μg/mL)d sample examined DOX (free) m-PEGMWCNTDOX E2-PEGMWCNTDOX

1

E2-PEGMWCNTDOX (ER−) E2-PEGMWCNTDOX (ER+)

2

a

lysosome

mitochondria

nucleus

0.197 ± 0.016

0.299 ± 0.023

0.62 ± 0.08

0.186 ± 0.012

0.274 ± 0.026

0.27 ± 0.09

− 98.9 ± 0.6

− 31.6 ± 0.2

2.4 ± 0.3a 1.6 ± 0.2b a***

2.3 ± 0.2a 1.5 ± 0.1b a***

99.3 ± 0.5

32.1 ± 0.1

1.0 ± 0.1 a***, b***

0.9 ± 0.1 a***, b***

A549

MCF 7

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

d

MWCNTs hardly influenced the loading or release characteristics of drug-loaded CNTs, compared to plain, oxidized MWCNTs or our recently reported HA-MWCNTs30 (Figure S6 of the Supporting Information). To investigate the cellular internalization and intranuclear releases of DOX, A549 cells were incubated with DOX@ E2-PEG-MWCNTs at 37 °C for 3 h. Confocal microscopy was done to visualize the released DOX/DOX@ f-CNTs. Figure 4A presents the confocal fluorescence image of single A549 cells incubated with DOX@ E2-PEG-MWCNTs. The micrographs (a−c) represent the DOX fluorescence, overlay of DOX fluorescence, and differential interference contrast (DIC) image of DOX@E2PEG-MWCNTs incubated cells. As evident from the images, the red fluorescence of DOX was highly accumulated in the nuclear and perinuclear region indicative of the presence of DOX/DOX-nanotubes inside as well as in the vicinity of the cell nuclei. To further validate our hypothesis, nuclear colocalization studies were also performed in a second ER (+ve) cell line (i.e., MCF 7) (Figure 4B). The nuclei of cells were stained with DAPI while DOX@f-CNTs was detectable by their intrinsic red fluorescence. In line with our expectation, both free DOX and DOX@m-PEG-MWCNTs presented little or practically no red fluorescence in the nuclei (Figure 4B, sections a and b). Contrastingly, DOX@E2-PEG-MWCNTs showed intense red fluorescence in the nucleus (Figure 4B, section c), as evident by a high Pearson colocalization coefficient (r > 0.6) value. The intranuclear delivery of DOX via E2-PEG-MWCNTs was further confirmed by alteration in nuclear morphology as evidenced by condensed nuclei. The appearance of condensed nuclei, an important hallmark of early apoptosis, was featured only in DOX@E2-PEG-MWCNTtreated cells (Figure 4C); no changes or abnormalities were

Pearson’s colocalization coefficient (r) formulation(s) tested

drug loading (%)c

c

Table 1. Pearson’s Colocalization Coefficient of Various fCNTs with Various Cell Organellesa S (no.)

entrapment efficiency (EE %)c

Data represents mean ± SEM (n = 3). G



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

observed in cells treated with either nontargeted conjugate/free drug (data not shown). 3.3. Anticancer Activity of DOX@E2-PEG-MWCNTs: A Correlation with Nuclear Targeting. To find any possible enhancement of anticancer efficiency of DOX@E2-PEGMWCNTs, both A549 and MCF 7 cells were incubated with free DOX, DOX@PEG-MWCNTs, and DOX@E2-PEGMWCNTs for 24 h. Cytotoxicity was evaluated by the MTT assay. The IC-50 values of free DOX and DOX@f-CNTs (normalized to DOX concentration) are summarized in Table 2. The order of cytotoxicity, as determined from the assay, was DOX@E2-PEG-MWCNTs > DOX@m-PEG-MWCNTs > DOX. It is, however, worthy to mention that DOX-deprived formulations presented little or practically no toxicity, even at concentrations greater than 100 μg/mL. To further validate the ER targeting capability of the synthesized conjugate, cytotoxicity study was also performed by pre-exposing the cells to excess (50 μg/mL) of E2, followed by incubation with DOX@ E2-PEG-MWCNTs. In the presence of E2, the cytotoxicity of the conjugate was significantly depreciated, and the observed growth inhibition (%) was even less than 10. To further validate the concept of nuclear targeting, A549 cells were incubated with free DOX, DOX@PEG-MWCNTs, and DOX@ E2-PEG-MWCNTs for 24 h, following which apoptosis and DNA fragmentation was quantified using TUNEL and DPA assays. As evident from Table 3, DOX@E2-PEG-MWCNTs

Table 3. Quantification of f-CNT Induced Apoptosis in A549 Cellsd apoptotic cells (%) sample examined control DOX (free) m-PEG-MWCNTDOX E2-PEG-MWCNTDOX

TUNEL assay

fragmented DNA (%) DPA assay

5.2 ± 1.5a 52.3 ± 5.2b a*** 56.2 ± 4.8c a***

1.98 ± 0.22a 31.3 ± 3.9 a*** 35.2 ± 3.1 a***

75.2 ± 6.8 a***, b*, c*

59.2 ± 5.5 a***, b*, c**

d Data represents mean ± SEM (n = 3). a*, b*, and c* represents quantification 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).

exhibited higher TUNEL (+ve) cells and fragmented DNA (75.2 ± 6.8%; 59.2 ± 5.5%) as compared to both nontargeted conjugate (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, intranuclear delivery of DOX through E2-PEG-MWCNTs could enhance the therapeutic efficacy of DOX in vivo. Subsequently, pharmacodynamic studies were carried out in chemically induced breast cancer using DMBA in female Sprague−Dawley (SD) rats. Animals were administered with 5 mg/kg of free H



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volume. The minimized tumor volume in rats after 10 days of treatment with DOX@E2-PEG-MWCNTs was approximately 18, 17, 5, and 2 times less as compared to rats treated with saline, drug-deprived E2-PEG-MWCNTs, free DOX, and DOX@m-PEG-MWCNTs, respectively. Representative photographs illustrating the reduction of the tumor burden in DOX@ E2-PEG-MWCNTs-treated rats have been presented as Figure 5B. The study, however, was terminated after 10 days, as no significant changes in tumor volume was observed in between 6 and 10 days. Consequently, animals were sacrificed on the 10th day and tumors were excised from sacrificed animals. Figure 5C presents the photographs of excised tumors treated with free DOX/DOX-fMWCNTs. While control and free DOX-treated tumor mass retained their original color, black carbon-based particles were uniformly distributed in the excised tumors pretreated with m-PEG-MWCNT-DOX and E 2 -PEGMWCNT-DOX. The intratumoral concentration of CNTs per unit mass of tumor homogenate was determined spectrophotometrically using an already standardized protocol described in our laboratory. As obvious, no traces of CNTs could be detected in either control- or DOX-treated groups. Interestingly, around 56.32 ± 4.67 and 78.26 ± 6.83 μg of CNTs per milligram of tumor homogenate were detected for m-PEG-MWCNT-DOX and E2-PEG-MWCNT-DOX, which corresponded to approximately 21.56% and 32.45% of the injected 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 free DOX and DOX-loaded CNT formulations for 15 days. After termination of the pharmacodynamic study on the 10th day, the various cardiotoxicity markers, including lactate dehydrogenase (LDH), cytokinin MB (CK-MB), and aspartate transaminase (AST) levels in plasma and superoxide dismutase (SOD) activity in heart homogenate were determined. An increase in LDH, CK-MB, or AST level and decreased SOD activity indicates cardiotoxicity. As evident from the various cardiotoxicity marker levels, DOX delivered through CNTs showed marked depreciation in cardiotoxicity in comparison to the free drug. As apparent from Figure 6 (panels a−b), following 10 days of treatment, the LDH level in heart tissue and CK-MB level in the plasma were significantly increased in the animal groups treated with free DOX. Conversely, LDH and CK-MB levels of various f-CNT-treated groups showed insignificant differences from the control group. In line with the results of other cardiotoxicity assessment parameters, AST level of free DOX treated rats showed significant elevation, indicative of myocardial infarction, whereas rats treated with f-CNTs presented insignificant differences from the control (Figure 6c). Concurrently, SOD levels in the heart homogenate of free DOX-treated group decreased while both DOX@m-PEGMWCNTs and DOX@E2-PEG-MWCNTs presented comparable SOD activity with respect to control (Figure 6d). In this connection, it may be noted that neither free DOX nor DOXloaded CNTs did present any detectable hepatoxicity or nephrotoxicity over the course of treatment.

DOX, DOX@m-PEG-MWCNTs, and DOX@E 2 -PEGMWCNTs (normalized to 5 mg/kg of free DOX). Figure 5A

Figure 5. (A) In vivo tumor growth inhibition profile of SD rats intravenously administered with free DOX and various functionalized MWCNT preparations loaded with an equivalent amount of the free drug. (B) Representative photograph of tumor reduction in rats treated with DOX@E2-PEG-MWCNTs over time. (C) Representative photograph and spectrophotometric data showing the intratumoral presence of DOX@f-MWCNTs.

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 rapid decline in tumor burden within 24 h of administration. Over the course of first 4 days’ treatment, both free DOX and DOXloaded CNTs led to a steady decrease in tumor burden in contrast to saline or plain E2-PEG-MWCNTs treated group showing continuous increase in tumor growth. On the sixth day, the minimized tumor volumes of rats treated with free DOX (87.36 ± 5.4%) differed significantly (p < 0.01) from DOX@m-PEG-MWCNTs (52.43 ± 3.65%) and DOX@E2PEG-MWCNTs (36.29 ± 3.45%). Thereafter, tumor volume of the free DOX-treated group started increasing while drugloaded CNTs maintained a constant reduction in the tumor

4. DISCUSSIONS In this study, E2-PEG-MWCNTs were synthesized by covalent coupling of amine-terminated E2-PEG with carboxyl-enriched MWCNTs. While both single- and multiwalled carbon nanotubes have sparked interest in their use as therapeutic vectors, we sought to proceed with MWCNTs because the I



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

membrane-associated and cytoplasmic ERs, which in turn, assisted their energy-dependent transport across the nuclear membrane (Figure 3C). Although the diameter of E2-PEGMWCNTs (30−50 nm) was too large to permeate across the nuclear pore (10−20 nm), steroids have been reported to dilate or perforate the nuclear pore (between 90 and 120 nm) through formation of nuclear−pore complexes (NPC)45.46 The transport of hydrophobic cargoes such as ER-E2-PEGMWCNTs (receptor−ligand) complex is believed to alter the permeability and conformational state of the NPCs, thereby initiating nuclear trafficking. At this point, it was necessary to examine the merits of nuclear-localized E2-PEG-MWCNTs in terms of their capability of intranuclear drug delivery and release. To have a proof-of-concept, DOX was loaded onto the sidewalls of m-PEG-MWCNTs and E2-PEG-MWCNTs, following which their cellular internalization and intranuclear release was investigated in two different ER (+ve) cell lines: A549 and MCF7. In case of DOX, very slight red fluorescence was visible either in the cell cytoplasm or the nucleus (Figure 4B, section a). Such observation is not against streamline because the overexpression of p-glycoprotein (Pgp) receptors on MCF 7 cells47 may limit the internalization of the free drug inside the cells. For m-PWG-MWCNT-DOX, red fluorescence was spread throughout the cell cytoplasm with very little red fluorescence in nuclei due to a concentration gradient diffusion mechanism of free DOX. Contrastingly, intense red fluorescence was visible in the nuclei of cells incubated with DOX@E2-PEG-MWCNTs. We inferred that free DOX or E2deprived DOX@m-PEG-MWCNTs cannot be directly delivered into the nuclei in the absence of any energy-dependent, receptor-mediated pathway. Likewise, DOX released from mPEG-MWCNTs in the cytoplasm is slowly diffused into the nucleus to exert therapeutic activity. However, as already shown in Figures 2 and 3, E2-PEG-MWCNT-conjugate presented the unique capability to localize in the cell nucleus, which enabled

latter presents a wider surface that permits a more efficient internal encapsulation and external functionalization with active molecules as compared to their single-walled counterparts.40 Further, in a number of studies focusing on nanotoxicology of CNTs, it has been found that MWCNTs are less toxic as compared to SWCNTs, which make them an ideal choice for in vivo applications.41,42 The successful immobilization of E2-PEG on the surface of carboxylated MWCNTs was substantiated through FT-IR and HRMAS-NMR analysis. The intracellular uptake of E2-PEG-MWCNTs by ER(+ve) MCF 7 cells was studied using confocal microscopy. As expected, E2-PEGMWCNTs presented appreciable internalization by its molecular target. To further apprehend the contribution of ERs in cellular internalization of E2-conjugated MWCNTs, competitive inhibition studies were performed. While E2pretreatment could not restrain CNTs from entering the cells, marked changes with regards to intracellular concentration of E2-PEG-MWCNTs was recorded. In this regard, it may be noted that although classical ERs are nucleocytoplasmic,43,44 some nonnuclear ER-αs are localized on the cell surface membrane as well. These membrane-bound ERs, combined with 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 selected A549 cells as our primary molecular target because this cell line presents a flat morphology, which is favored for intracellular colocalization analysis. Our results showed that E2-PEGMWCNTs had nominal proclivity to accumulate in lysosome or mitochondria but significant propensity toward nuclear compartmentalization. These results strengthened our expectations regarding the active intervention of ERs in the intracellular uptake as well as the nuclear localization of drugloaded nanotubes. It seemed that the presence of E2 on the surface of CNTs facilitated the binding of the conjugate with J



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(EPR) effect of the hydrophilic PEG spacer interconnecting E2 and MWCNTs (passive targeting), (ii) intrinsic cell penetrability of MWCNTs via the well-established “nanoneedle pathway”, and (iii) the slow, sustained intranuclear drug delivery via an ER-mediated pathway. Direct transport of the chemotherapeutic agent to nucleus is not possible in the case of E2-deprived PEGylated MWCNTs so that antitumor efficacy of the conjugate is somewhat compromised and is twice less than its targeted counterpart. These results suggest that combination of MWCNTs, PEG, E2, and DOX into a single system results in a synergistic anticancer response, justifying the rationale of our carrier design. While free DOX treatment induced severe cardiotoxicity in animals, animals treated with DOX@m-PEGMWCNTs and DOX@E2-PEG-MWCNTs were devoid of any detectable hepatotoxicity, nephrotoxicity, and cardiotoxicity. Cumulatively, these results suggest that the delivery property of MWCNTs was effectively combined with ER avidity of E2 to facilitate intranuclear drug delivery to breast tumors, while mitigating drug as well as carrier-associated toxicity.

the conjugate to directly release the loaded drug at the nucleus itself. It can also be known that the red fluorescence in Figure 4B (section c) is from DOX@E2-PEG-MWCNT-conjugate rather than that of free DOX that had already been released in the cytosol and then diffused into nuclei. The above observations are more qualitatively confirmed by the scattered plot of fluorescent intensity of selected MCF7 cells, as shown in Figure 4B. The intranuclear localization of DOX@E2-PEGMWCNTs ensures slow, sustained release of DOX specifically to its pharmacodynamic site of action so that hallmarks of apoptosis (viz. condensed nuclei) were visible as early as 3 h (Figure 4C). In attempts to find the possible enhancements in anticancer efficacy by E2-PEG-MWCNTs, we observed that DOX-loaded MWCNTs, in general, exerted higher cytotoxicity compared to free DOX (Table 1). The higher cytotoxicity of DOX@mPEG-CNTs compared to free DOX may be accounted to higher intracellular availability of DOX, delivered through an intrinsically cell-penetrable carrier like CNTs. Between plain DOX@m-PEG-MWCNTs and DOX@E2-PEGMWCNTs, the IC-50 of the ER-targeted conjugate in A549 and MCF 7 cells was 1.5−1.6 times lower than its nontargeted counterpart (P < 0.001) which, however, seems to be a consequence of direct, intranuclear delivery of DOX intervened by E2-PEG-MWCNTs that was not operative in the case of the nontargeted conjugate. Depreciation in cytotoxicity in the case of E2 pretreatment revalidates that the presence of E2 on the surface of MWCNTs play a key role in targeting DOX to its site of action (i.e., nucleus). This hypothesis was revalidated from apoptosis studies in which DOX@m-PEG-MWCNTs showed higher TUNEL(+ve) cells (a hallmark of late apoptosis) and fragmented DNA compared to both free drug and nontargeted control. Having established the merits of our newly synthesized carrier system with regards to cell-selective, intranuclear drug delivery, we tried to understand whether the effect can be translated into an equivalent therapeutic response in vivo. In line with that approach, pharmacodynamic studies were conducted in chemically, breast tumor-induced rats. DMBA was used as the carcinogen. We opted to work on this particular model because DMBA is widely used to induce breast tumors in rodents with the average rate of tumor induction varying from 65 to 75%. Finally, there have been reported evidence of estrogen receptor overexpression in DMBA-derived mammary tumors in female SD rats.48 In this regard, it is worth noting that 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 cytotoxicity experiments. In addition, intratumoral presence of CNTs at the tumor site was confirmed through spectrophotometric analysis of f-CNTs in the tumor homogenate, which was further supported through visual observations. The higher intratumoral concentration of DOX@E2-PEG-MWCNTs over DOX@mPEG-MWCNTs may be attributed to its prolonged intratumoral presence, facilitated through ligand−receptor binding interaction. As ERs are overexpressed in more than 80% of breast tumors, the estradiol moiety present on the surface of MWCNTs can potentially interact and bind with cytoplasmic ERs. This interaction protracts the intracellular retention of drug-loaded nanotubes. The higher antitumor activity of DOX@E 2 -PEG-MWCNTs compared to DOX@m-PEGMWCNTs or free DOX may be attributed to their high intratumoral accumulation and prolonged intracellular availability, facilitated by (i) enhanced permeability and retention

5. CONCLUSION In conclusion, we have prepared a novel, E2-PEG-MWCNT conjugate that can selectively target ER overexpressing cancer cells and facilitate direct, intranuclear drug delivery through an ER-dependent pathway. By loading DOX onto the sidewalls of E2-PEG-MWCNTs, we demonstrated that conjugation of E2 with MWCNTs not only augmented the nuclear targeting index of carrier-bound drug but also synergized its therapeutic efficacy both in vitro and in vivo while alleviating drugassociated cardiotoxicity. The therapeutic conjugate was further integrated with an organic fluorophore viz. AF-647/RITC to enable real-time monitoring of intracellular, organelle-specific localization of the targeted nanoprobe through optical fluorescence imaging. For the first time, we have embarked on the feasibility of cancer cell-selective, nuclear-targeted drug delivery using steroid-conjugated MWCNTs as the molecular transporter. Although further studies are required to elucidate the detailed mechanism of nuclear penetration and long-term fate of E2-PEG-MWCNTs in vivo, proof-of-concept realized from the present study can be extended to augment the second- and third-order targeting of a myriad of antineoplastic drugs, which exert their pharmacodynamic action on the nucleus.

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ASSOCIATED CONTENT

S Supporting Information *

FT-IR, MALDI-TOF and HRMAS-NMR mass spectra, SEM and TEM images, confocal images, and drug release profile. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], sanyogjain@rediffmail.com. Tel: +91172-2292055. Fax: +91172-2214692. Notes

The authors declare no competing financial interest.

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



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grateful to the Department of Science and Technology (DST), GOI, New Delhi for providing postdoctoral and senior research fellowships, respectively. Director NIPER is acknowledged for providing necessary infrastructural facilities. Thanks are due to Dr. 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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