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Colloids and Surfaces B: Biointerfaces 132 (2015) 17–26

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

j o ur nal ho me pa ge: www.elsev ier .com/ locate /co lsur fb

ancer targeting propensity of folate conjugated surface engineeredulti-walled carbon nanotubes

eelesh Kumar Mehraa,b, N.K. Jaina,b,∗

Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar 470 003, IndiaPharmaceutical Nanotechnology Research Laboratory, ISF College of Pharmacy, Moga 142 001, India

r t i c l e i n f o

rticle history:eceived 4 November 2014eceived in revised form 25 April 2015ccepted 27 April 2015vailable online 7 May 2015

eywords:arbon nanotubesocetaxelharmacokinetic

a b s t r a c t

Our main aim in the present investigation was to investigate the cancer targeting potential of docetaxel(DTX) loaded, folic acid (FA) terminated, poly (ethylene glycol) (PEG) conjugated, surface engineeredmulti walled carbon nanotubes (DTX/FA-PEG-MWCNTs) in tumor bearing Balb/c mice. The percent load-ing efficiency of DTX/FA-PEG-MWCNTs and DTX loaded MWCNTS (DTX/MWCNTs) was calculated tobe 93.40 ± 3.82% and 76.30 ± 2.62%, respectively. Flow cytometry analysis suggested that the DTX/FA-PEG-MWCNTs arrested MCF-7 cells’ cycle in the G2 phase and was more cytotoxic as compared toDTX/MWCNTs as well as free drug solution. The obtained pharmacokinetic parameters clearly describethe biocompatibility of engineered nanotubes to degree of functionalization and ability for prolongedresidence inside the body. DTX/FA-PEG-MWCNTs was found to be significantly more efficient in tumor

aplan–Meier survivalumor growth inhibitionnticancer activity

suppression as compared with plain MWCNTs (non-targeted) as well as drug solution owing to theenhanced drug release from endosomes after internalization. The DTX/FA-PEG-MWCNTs showed highlysignificant prolonged survival span (40 days) as compared to DTX/MWCNTs (24 days), free DTX (19 days)and control group (12 days). Overall, we can conclude that the DTX/FA-PEG-MWCNTs shows highercancer targeting propensity vis a vis minimal side effects in tumor bearing Balb/c mice.

© 2015 Elsevier B.V. All rights reserved.

. Introduction

The development of ‘safe and effective’ nanomedicines forhe treatment of diseases including diabetes, acquired immuneeficiency syndrome, tuberculosis and cancer still remains the fore-ost challenging task to researchers, scientists and academicians,orldwide. Cancer accounted for 7.6 million deaths (approximately

3% of all deaths) in 2008 according to recent Fact Sheet of Worldealth Organization (WHO) wherein approximately 70% deathsccurred in low- and middle-income countries. In 2030, cancereaths are projected to rise over 13.1 million, worldwide [1]. Theailure of chemotherapy is due to the non-selectivity as well asnability to target the anticancer agent(s) to the cancerous cells.

he various available nano-sized carrier systems including den-rimers [2], nanoparticles [2] and carbon nanotubes [3–8] are being

∗ Corresponding author at: Pharmaceutics Research Laboratory, Departmentf Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar 470 003, India.el.: +91 7582 265055; fax: +91 7582 265055.

E-mail addresses: neelesh81mph@gmail.com (N.K. Mehra),narendr@yahoo.co.in (N.K. Jain).

ttp://dx.doi.org/10.1016/j.colsurfb.2015.04.056927-7765/© 2015 Elsevier B.V. All rights reserved.

continuously explored for improved specificity and targeting aswell as realizing the attributes of the ‘magic bullet’ concept.

In the past two decades, surface engineered carbon nanotubes(CNTs) have been explored designed and considered as valuable,promising, ‘safe and effective’ alternative nano-architecture forpharmaceutical and biomedical applications due to their uniquephysicochemical properties. CNTs comprise of thin graphite sheetsof condensed benzene rings rolled upon into the nanoneedle, seam-less tubular hollow cylinder. CNTs can be distinguished on the basisof their lengths, diameters, and most importantly, presence of wallsand are categorized into single-, double-, triple-, and multi-walledcarbon nanotubes [3,9–11]. The pristine CNTs (first generation;untreated CNTs) are not suitable for drug delivery on account oftheir hydrophobicity and toxicity due to the presence of impurities,which can fortunately be overcome by surface functionalization.Higher degree of functionalization (hence lower toxicity) makesnanotubes better, safer and effective drug delivery system [12].

The surface alterations of CNTs can be performed either by cova-lent or non-covalent interactions depending on the intermolecular

interaction. The non-covalent modifications, based on the extended�-system (p-orbital) of the nanotubes sidewall, interact with theguest chemical moieties through �–� stacking interactions. Cur-rently, surface engineered CNTs are being explored for targeted

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elivery and have been claimed to be non-toxic to human cells9,12,13].

Docetaxel (N-debenzoyl-N-tert-butoxycarbonyl-10-deacetyl-aclitaxel) used in the present investigation is a semi-syntheticaxane, derived from the precursor 10-deacetyl baccatin III andxtracted from the European yew tree Taxus baccata for targetingo �-subunit of tubulin. It entered into clinical trials in 1990 andemonstrates the efficacy in the treatment of several malignancies

ncluding prostate, small and non-small cell lung cancer and breastancer etc. [14].

In the present investigation we intended to explore the can-er targeting potential of the docetaxel (DTX) bearing surfacengineered MWCNTs. The developed DTX bearing surface engi-eered MWCNTs nanoconjugates were characterized for loadingfficiency, in vitro release, hemocompatibility and toxicity in tumorearing Balb/c mice.

. Experimental

.1. Materials

Multi Walled Carbon Nanotubes (MWCNTs) produced by chem-cal vapor deposition (CVD) with 99.3% purity, were purchased fromigma Aldrich Pvt. Ltd. (St. Louis, Missouri, USA). Docetaxel waseceived as a benevolent gift from M/s Fresenius Kabi Oncology Ltd;FKOL) (formerly Dabur Research foundation), Sahibabad, India. Alleagents and solvents were used as received.

.2. Surface engineering of the pristine MWCNTs

Firstly, the procured pristine MWCNTs were purified by treat-ng in a microwave oven (GEM Insta Cook, Gurgaon, India) at00 ± 2 ◦C for 2 h. The microwave treated MWCNTs (500 mg) wereefluxed with a mixture of concentrated Nitric and Sulphuric acidHNO3:H2SO4::1:3 ratio) in a flat bottom flask (equipped withhe reflux condenser and thermometer) with continuous mag-etic stirring (100 RPM; Remi, Mumbai, India) at 120 ± 5 ◦C for

h; washed, ultra centrifuged (20,000 rpm for 15 min; Z36HK,ERMLE LaborTchnik GmbH, Germany), vacuum dried (Jyoti Sci-ntific Industries, Gwalior, India), lyophilized (Heto dry Winner,enmark, Germany), and collected [9,13,15].

.3. Folic acid (FA) conjugation with surface engineered MWCNTs

The MWCNTs were conjugated with folic acid (FA) using PEGpacer and characterized following the method reported by us ear-ier [9].

.4. Loading efficiency

The MWCNTs: DTX in optimized ratio (1:2) were added tonhydrous ethanol (0.5 mL) in an ultrasonic bath for about 15 minith drop-wise addition of PBS (pH 7.4) solution and ultrasoni-

ated using an ultrasonic probe (400 W) for approximately 10 minLark, Chennai, India). The resultant suspension was ultracen-rifuged at 10,000 rpm for 10 min until the MWCNTs were fullyeparated, and the obtained supernatants were discarded. Theemaining solids were thoroughly rinsed with anhydrous ethanolnd deionized water to remove excess docetaxel. The amount ofnbound DTX in the solution was determined by measuring thebsorbance at �max 230 nm in a spectrophotometer (Shimadzu601, UV–Visible Spectrophotometer, Shimadzu, Japan) and the

TX loading efficiency was calculated (n = 3). The product was col-

ected, dried, lyophilized (Heto dry winner, Denmark, Germany)nd stored at 5 ± 3 ◦C for further studies. Finally, the DTX loadedWCNTs formulations i.e. DTX loaded MWCNTs (DTX/MWCNTs)

s B: Biointerfaces 132 (2015) 17–26

and DTX loaded FA-PEG-MWCNTs (DTX/FA-PEG-MWCNTs) wereprepared.

2.5. Characterization of pristine and functionalized MWCNTs

The pristine and surface engineered MWCNTs were extensivelycharacterized using different analytical characterization tools. Thesurface topography of the pristine and surface engineered MWC-NTs was determined through transmission electron microscopy(TEM; Morgagni 268-D, Fei Electron Optics, Holland) after dryingon carbon-coated copper grid and negative staining with 1% phos-photungstic acid (PTA) [13].

The surface fracture of the nanotubes nanoformulations wasstudied using atomic force microscopy (AFM) in a tapping modewith Digital Nanoscope IV Bioscope (Veeco Innova Instruments,Santa Barbara, CA, USA) after drying in air.

The average particle size and size distribution were determinedby photon correlation spectroscopy in a Malvern Zetasizer nanoZS90 (Malvern Instruments, Ltd, Malvern, UK) at room temperature(RT) after addition of surfactant.

The Raman spectra of the pristine and f-MWCNTs fororder-disorder hexagonal carbon were recorded using Ramanmicro-spectroscopy RINSHAW, inVia Raman Spectrophotometer(RENISHAW, Gloucestershire, UK). The microspectrophotometerwas operated with 532 nm laser radiation under objective lens of20× magnification (Olympus BX 41, USA) with a slit of 1 × 6 mmwhereas the incident power was approximately 1 mW with 30 sexposure time [13].

The X-ray diffractograms (XRD) were recorded (X-ray diffrac-tometer, PW 1710 Rigaku, San Jose, CA) by adjusting X-ray powerof 40 kV and 40 mA of MWCNTs formulations [13].

2.5.1. In vitro release studiesThe release of docetaxel from the developed MWCNTs formu-

lations (DTX/FA-PEG-MWCNTs and DTX/MWCNT) was monitoredseparately in sodium acetate buffer saline pH 5.3 (lysosomal pH),and phosphate buffer saline pH 7.4 (physiological pH) througha modified dialysis diffusion technique while maintaining thephysiological temperature 37 ± 0.5 ◦C throughout the study (n = 3)[13,14,16–19]. The known amount of DTX loaded MWCNTs for-mulations (10 mL) was added in the dialysis sac (MWCO, 12 kDa),hermetically tied and placed into the receptor compartment(ethanol: phosphate buffer pH 5.3:7.4::3:7 containing Tween 80)with slow and continuous magnetic stirring at 37 ± 0.5 ◦C understrict sink condition. Tween 80 was used in the release medium tosolubilize the DTX and to facilitate the passage across the dialysismembrane. Aliquots were withdrawn at definite time points fromthe mixture and immediately replenished with an equal volumeof fresh medium for estimation of the concentration of DTX usingUV/Visible spectrophotometer at �max 230.0 nm (UV/Vis, Shimadzu1601, Kyoto, Japan).

2.5.2. Accelerated stability studyThe DTX/FA-PEG-MWCNTs and DTX/MWCNTs were stored in

tightly closed glass vials separately in dark as well as in amber col-ored and colorless glass vials at 5 ± 3, 25 ± 2 and 40 ± 2 ◦C for aperiod of six months in stability chambers (Remi CHM-6S, India)(n = 3) [13,20]. The MWCNTs formulations were analyzed initiallyand periodically up to six months for any change in particle size,drug content and organoleptic features like aggregation, precipita-tion, color and odor, if any.

2.6. Comparison of hemolytic toxicity

The hemolytic toxicity of the administered MWCNTs formula-tions was assessed in vitro [13,19]. Briefly, fresh whole human blood

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as collected in Hi-Anticlot blood collecting vials (HiMedia, Mum-ai, India) and centrifuged at 3000 rpm (Remi, Mumbai, India) for5 min in an ultracentrifuge (Z36HK, HERMLE LaborTchnik GmbH,ermany). The red blood corpuscles (RBCs) were collected from

he bottom and separated out, washed with physiological normalaline (0.9%; w/v) until clear and the colorless supernatant wasbtained above the cell mass. The RBCs suspension (1 mL) wasixed separately with 0.9% (w/v) normal saline (4.5 mL), free DTX,TX/MWCNTs and DTX/FA-PEG-MWCNTs dispersions (0.5 mL) and

ncubated for 60 min, to allow interaction. After incubation, sam-les were centrifuged for 15 min at 1500 rpm and the supernatantas taken to quantify the hemoglobin content at �max 540 nm

pectrophotometrically considering 0.9% w/v NaCl solution (nor-al saline) and deionized water, respectively as zero and 100%

emolytic (n = 3).

.7. Cell culture experiment

The MCF-7 (Michigan Cancer Foundation; folate overexpress-on) derived from pleural effusion was procured from Nationalenter for Cell Sciences (NCCS), Pune, India. Before starting cellulture experiments, the cells were pre-cultured until approxi-ately 80% confluence was reached. The cell lines were cultured

n Dulbecco’s Modified Eagle Medium (DMEM; HiMedia, Mum-ai, India) containing 10% fetal bovine serum (FBS; HiMedia,umbai, India) supplemented with 2 mM l-glutamine and 1%

enicillin–streptomycin mixture (Sigma, St Louis, Missouri) inumidified atmosphere containing 5% CO2 at 37 ◦C [13].

.7.1. Methylthiazole tetrazolium (MTT) cytotoxicity assayThe MCF-7 cells were incubated in 96-well transparent tissue

ulture plates at density of 1 × 104 cells/well. After 12 h the oldedium was removed and cells were incubated separately with theTX, DTX/MWCNTs and DTX/FA-PEG-MWCNTs at equivalent DTXoncentrations (0.001, 0.01, 1, 10, 100 �M) and allowed to adhereor 24 and 48 h at 37 ◦C prior to assay. The medium was decantednd 50 �L of methylthiazole tetrazolium (MTT) (1 mg/mL) in DMEM10 �L; 5 mg/mL in Hank’s balanced Salt Solution; without phenoled) was added and incubated. The experiment was performed inriplicate. The absorbance of the wells was measured at 570 nm andhe percent cell viability was calculated using following formula:

ell viability (%) = [A]test

[A]control× 100

here [A]test is the absorbance of the test sample and [A]control ishe absorbance of control samples.

.7.2. Cell cycle analysisCell cycle distribution of the developed nanotubes formula-

ions in different phases was studied using the cultured MCF-7ells (n = 3). Briefly, the cultured MCF-7 cells were treated withhe developed MWCNTs formulations (2 nM/mL) for 24 h whilehe cells treated with culture medium served as control. The cellsere harvested by centrifugation, washed with ice-cold PBS andxed using 70% cold ethanol overnight. Subsequently cells wererypsinized, washed and fixed in 70% ethanol and further treatedith Ribonuclease A (DNase free, 100 �g/mL) and propidium iodide

PI; 50 �g/mL) for 30 min at 37 ◦C in dark. The treated cells wereentrifuged and obtained cell pellets were re-suspended with PBS

nd kept until use. The percent of cells arrested in different phasesG1, G2, and S phase) of the cell cycle event was counted usingycle analysis software with FACSCalibur Flow Cytometer (Becton,ickinson Systems, FACS cantoTM, USA) [13,21–23].

s B: Biointerfaces 132 (2015) 17–26 19

2.7.3. Cellular uptake of the formulations through flow cytometryThe cellular uptake study of free DTX and DTX loaded MWCNTs

formulations labeled in 5:1 ratio of DTX with fluorescein isothio-cyanate (FITC) solution (100 �g/mL in DMSO) using MCF-7 cell linewas performed in a flow cytometer for quantitative analysis (n = 3).The developed formulations and free DTX were incubated for 4 has in case of DNA cell cycle content. After 3 h exposure, cells werewashed with cold-PBS, trypsinized, centrifuged and analyzed quan-titatively using FACSCalibur Flow Cytometer (Becton, DickinsonSystems, FACS cantoTM, USA).

2.8. In vivo studies

The in vivo studies were carried out with prior approval ofInstitutional Animal Ethics Committee as per guidelines of the Com-mittee for the Purpose of Control and Supervision of Experimentson Animals (CPCSEA) of Dr. H.S. Gour Vishwavidyalaya, Sagar (M.P.),India (Registration No. 379/01/ab/CPCSEA/02). The Balb/c mice ofuniform weight (20–25 g) were housed in ventilated plastic cagesand allowed access to water ad libitum. The Balb/c mice were fed ona special low-folate diet and acclimatized at 25 ± 2 ◦C and 50–60%relative humidity under natural light/dark condition prior to in vivostudy [9].

The tumor was developed using right flank method by injectingserum-free cultured MCF-7 cells (1 × 107 cells) in the right hind legof the mice. The tumor development was monitored by palpatingthe injected area with index finger and thumb for the presence ofthe tumor (approximately 100 mm3) [9,13,24].

2.8.1. Determination of pharmacokinetic parameters afterintravenous administration

The pharmacokinetic parameters after intravenous (i.v.) admin-istration of free DTX and DTX loaded nanotubes formulations(30 mg/kg body weight) were determined. The blood samples werecollected in Hi-Anticlot blood collecting vials (HiMedia, Mumbai,India) at different time points (0.25, 0.5, 1, 2, 3, 6, 12, 18, 24 and48 h) from the retro-orbital plexus of eyes (animals) under mildanesthesia. The supernatant (serum) was collected after centrifu-gation of blood, vortexed and ultracentrifuged (Z36HK, HERMLELaborTchnik GmbH, Germany), and finally the concentration ofdrug was determined by High Performance Liquid Chromatography(HPLC) method and different pharmacokinetic parameters werecalculated.

2.8.2. Tissue/organ biodistribution studyThe organ biodistribution study of the free DTX, DTX/MWCNTs

and DTX/FA-PEG-MWCNTs formulations was performed on tumorbearing Balb/c mice (n = 3). The formulations (equivalent dose ofDTX = 30.0 mg/kg body weight) were sterilized using 0.2 �m milli-pore filter and administered intravenously through caudal tail veinroute. The mice were carefully sacrificed by decapitation at 1, 6, 12and 24 h time points for the collection of organs like liver, spleen,kidney, heart, and tumor. The collected organs were washed inRinger’s solution, dried with the help of tissue paper, weighed andstored frozen till used. The required quantity of ethanol was addedand homogenized (York Scientific Instrument, New Delhi, India),vortexed and ultracentrifuged at 3000 rpm for 15 min (Z36HK,HERMLE LaborTchnik GmbH, Germany). The clear supernatant wascollected, injected into an HPLC system (Shimadzu, C18, Japan) andassayed for DTX content wherein mobile phase consisted of ace-tonitrile:methanol:0.02 M ammonium acetate buffer (pH 5.0) in20:50:30; v/v/v ratio at 1 mL/min flow rate at 102/101 bar pressure.

2.8.3. Assessment of anti-tumor targeting efficacyThe in vivo cancer targeting efficacy of the DTX/FA-PEG-

MWCNTs and DTX/MWCNTs formulations was determined in

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umor bearing Balb/c mice. The Balb/c mice were accommodated in pathogen-free laboratory environment during the tenure of thetudies. Tumor measurement was performed using electronic digi-al Vernier Caliper and the tumor volume at the longest and a widestwo dimension point was measured. The tumor size was calculatedsing the formula = 1/2 × length × width2 and median survival timeas also recorded. The in vivo tumor study was terminated 45 daysost-treatment [13].

The hematological study was performed following a reportedethod for DTX/FA-PEG-MWCNTs, DTX/MWCNTs and free DTX

nd analyzed at a local pathology laboratory [9,13,25].

.9. Statistical analysis

The results were expressed as mean ± standard deviation (n = 3)nd statistical analysis was performed with Graph Pad Instat Soft-are (Version 3.00, Graph Pad Software, San Diego, California, USA)

y one-way ANOVA followed by Tukey–Kramer test for multipleomparison. The pharmacokinetic data analysis of plasma concen-ration time profile was conducted using the Kinetica softwareThermo scientific, USA) followed by non-compartment analysis.

probability of p ≤ 0.05 was considered significant while p ≤ 0.001as considered to be extremely significant.

. Results and discussion

Folic acid (FA), also known as folate, vitamin Bc (folacin), vitamin9, M, pteroyl-l-glutamic acid, is a water-soluble vitamin, neces-ary for the synthesis of purines and pyrimidines. FA conjugatedanocarriers are known to exhibit ligand–receptor interactions,

nternalized through caveolae-mediated endocytosis mechanism,nd release the drug molecules into the cytoplasm. Numeroustudy reports are already available on folate-mediated targetingf anticancer bioactives [9,16,21,26,27]. Castilo et al. reported theon-covalent conjugate of SWCNTs and FA aimed to interact withells over-expressing folate receptors using rapid ‘one pot’ syn-hesis method. The low toxicity of SWCNTs-FA by cancer cellsuggested their potential use in drug delivery and diagnosis of can-er or treatment of tropical diseases such as leishmaniasis [28].n the current scenario, surface tailored carbon nanotubes (CNTs)re attracting great attention in the treatment of cancer including

heragnostic applications. We have initially functionalized the pro-ured MWCNTs with PEG spacer and appended the folic acid (FA) as

targeting ligand for specific targeting [9]. The high drug loadingbility of the surface engineered MWCNTs suggests the potential

Fig. 1. Transmission electron microscopic image of (A) DTX/FA-PEG-MWCNTs, (B) DT

s B: Biointerfaces 132 (2015) 17–26

application of CNTs as a targeted drug delivery system. The percentloading efficiency of DTX in DTX/MWCNTs and DTX/FA-PEG-MWCNTs formulations determined through modified dissolutionmethod was found to be 76.30 ± 2.62% and 93.40 ± 3.82%, respec-tively. This high percent loading efficiency was achieved due tostrong hydrophobic, electrostatic and �–� stacking interactionsamong CNTs and DTX. The high loading efficiency of engineerednanotubes makes it a better carrier with better stability at normalpH and sustained release in acidic microenvironments (lower pH).Our in vitro results are in good agreement with previous reports[19,28,29].

The TEM and AFM were used to investigate the surfacemorphology in terms of size, shape and topography of the devel-oped engineered MWCNTs formulations. TEM photomicrographssuggest that the nanotube formulations were tubular and in nano-metric size range (Fig. 1A and B). AFM analysis also revealed thenanoneedle tubular structure of the DTX/FA-PEG-MWCNTs formu-lation (Fig. 1C).

The average particle size (nm) and size distribution (PSD)with polydispersity index (PDI) were determined in a MalvernZetasizer nano ZS90 (Malvern Instruments, Ltd, Malvern, UK) atroom temperature (RT). The particle size of the DTX/MWCNTsand DTX/FA-PEG-MWCNTs was found to be 220.41 ± 9.50 (PI-0.27 ± 0.02) and 240.28 ± 8.60 nm (PI-0.42 ± 0.06), respectively.Ren and co-workers reported the particle size and polydisper-sity index (PI) of the doxorubicin loaded angiopep-2 modifiedPEGylated oxidized MWCNTs (DOX-O-MWCNTs-PEG-ANG) to be202.23 ± 3.43 nm and 0.342 ± 0.01, respectively [24].

The XRD analysis of functionalized MWCNTs and FA-PEG-MWCNTs clearly precludes any change in the original seamlesstubular structure as in case of pristine MWCNTs (Fig. 2A and B).

Raman spectroscopy provides information about the hybridiza-tion state and the defect chemistry of the CNTs. CNTs have four mainbands in Raman spectrum (i) radial breathing mode (RBM), (ii) G-band, (iii) D-band, and (iv) D′ mode [13,29]. The Raman spectrumof the purified MWCNTs showed the Raman shift at 1579.85 cm−1

and at 1346.15 cm−1, which correspond to the G band (graphite-like mode) and D band (disorder-induced band), respectively. TheRaman spectrum of the DTX/FA-PEG-MWCNTs showed the G bandaround 1565 cm−1 and D band around 1310 cm−1. The shifting ofthe G and D band to the lower Raman intensity in the DTX/FA-PEG-

MWCNTs is mainly due to increase in the extension of conjugationof FA with functionalized MWCNTs, which would increasethe single bond characteristic in the functionalized systems(Fig. 2C and D).

X/MWCNTs and (C) atomic force microscopic image of DTX/FA-PEG-MWCNTs.

N.K. Mehra, N.K. Jain / Colloids and Surfaces B: Biointerfaces 132 (2015) 17–26 21

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Fig. 2. X-ray diffraction pattern of (A) pristine, and (B) DTX/FA-PEG-MWCN

The cumulative in vitro release of DTX from the surface tailoredWCNTs formulations was studied at the normal physiological

nd lysosomal pH for determining the overall pharmaceutical ther-peutic efficacy in blood stream and at target site (Fig. 3). TheH of the cytosol is neutral to mildly alkaline (7.4–7.8) while

ysosomal pH is acidic (4–5.5). During the internalization of theTX/MWCNTs into the target MCF-7 cells, initially the drug has

o be released from the nanotubes formulations in order to exertts overall therapeutic effect. The in vitro release behavior ofTX from the surface engineered MWCNTs formulations exhib-

ted biphasic pattern that was characterized by an initial fasterollowed by sustained release. At pH 5.3 and 7.4 the cumulativeercent DTX release was found to be 93.20 ± 3.76, 33.20 ± 1.88nd 85.90 ± 3.82, 17.40 ± 0.10 for DTX/MWCNTs, and DTX/FA-PEG-WCNTs, respectively in 24 h whereas in 200 h cumulative DTX

elease from DTX/FA-PEG-MWCNTs was found to be 70.22 ± 3.02nd 54.60 ± 1.45 at pH 5.3 and 7.4, respectively. The sustainedelease of DTX was observed due to the limited solubility andtrong hydrophobic interaction among DTX and surface engineered

WCNTs. Arora and co-workers reported the development ofWCNTs-docetaxel conjugates by covalent interaction, involving

ucleophilic substitution reaction mechanism and reported that

ig. 3. Cumulative amount of DTX released from the DTX/MWCNTs and DTX/FA-PEG-MWValues represented as means ± SD; n = 3).

d Raman spectra of (C) pristine, and (D) DTX/FA-PEG-MWCNTs conjugate.

the drug release from the docetaxel-MWCNTs conjugate was fasterin acidic pH, as compared to that in buffer of normal cell pH [29].Our results are in good agreement with previous reports [29,31,32].

The developed nanotube formulations were found to be moststable in dark at 5 ± 3 ◦C. However, on storage in light at 25 ± 2 ◦C,slight turbidity was observed, which might be due to aggregationof nanotubes (Tables 1 and 2). At 40 ± 2 ◦C, all the formulationsshowed higher turbidity that may be ascribed to the formation oflarger aggregates and bundling of nanotubes [20]. The drug leakagefrom the developed nanotubes formulations is another importantparameter, which was measured to assess the stability. The drugleakage from the developed nanotube formulations was found to benegligible at 5 ± 3 ◦C, hence considered being most stable at 5 ± 3 ◦Ctemperature in dark condition. Our results are in good agreementwith previous reports [9,30]. The percent hemolysis of pristineMWCNTs (18.0 ± 0.50%), oxidized MWCNTs (15.50 ± 0.56%), DTX(19.20 ± 0.45%), DTX/MWCNTs (14.87 ± 0.44%) and DTX/FA-PEG-MWCNTs (9.20 ± 0.14%) was determined on collected whole humanblood. Pristine MWCNTs showed highest (18.0 ± 0.50%), while

DTX/FA-PEG-MWCNTs showed minimum (9.20 ± 0.14%) hemolytictoxicity. The pristine nanotubes exhibit high hemolytic toxicity dueto their inherent toxicity while surface engineering of MWNCTs

CNTs nanoconjugates at 37 ± 0.5 ◦C in phosphate buffer solution (pH = 5.3 and 7.4).

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Table 1Accelerated stability studies for the DTX/MWCNTs formulations.

Stability parameter DTX/MWCNTs after 6 months

Dark Light

T1 T2 T3 T1 T2 T3

Turbidity − − ++ + ++ +++Precipitation − − ++ + ++ ++Change in color − − + + + ++Crystallization − − + + + ++Change in consistency − − + + + ++Percent drug leakage (after months)1 1.40 ± 0.02 1.80 ± 0.03 2.62 ± 0.04 2.90 ± 0.05 3.51 ± 0.04 8.83 ± 0.442 2.82 ± 0.03 2.21 ± 0.03 4.62 ± 0.04 3.80 ± 0.04 5.02 ± 0.03 10.85 ± 0.044 3.81 ± 0.06 3.02 ± 0.04 5.03 ± 0.04 5.62 ± 0.02 7.83 ± 0.02 13.47 ± 0.026 5.02 ± 0.02 4.61 ± 0.08 5.42 ± 0.06 7.82 ± 0.07 9.64 ± 0.07 15.63 ± 0.08

Table 2Accelerated stability studies for the DTX/FA-PEG-MWCNTs formulations.

Stability parameter DTX/FA-PEG-MWCNTs after 6 months

Dark Light

T1 T2 T3 T1 T2 T3

Turbidity − − ++ + ++ +++Precipitation − − ++ + ++ ++Change in color − − + + + ++Crystallization − − + + + ++Change in consistency − − + + + ++Percent drug leakage (after months)1 0.40 ± 0.02 1.41 ± 0.05 1.92 ± 0.04 1.42 ± 0.03 1.80 ± 0.02 5.71 ± 0.062 1.10 ± 0.03 1.80 ± 0.04 2.91 ± 0.05 1.73 ± 0.07 2.21 ± 0.08 6.02 ± 0.024 1.90 ± 0.03 2.10 ± 0.07 3.30 ± 0.07 2.21 ± 0.04 3.00 ± 0.03 6.51 ± 0.066 2.10 ± 0.05 2.50 ± 0.06 3.81 ± 0.04 2.60 ± 0.09 3.42 ± 0.08 7.33 ± 0.03

T es repc

aicPs

ficmincAt

F(

1, T2 and T3 represent 5 ± 3, 25 ± 2, and 40 ± 2 ◦C temperatures, respectively. Valuonsiderable change and major change, respectively.

nd conjugation of FA-PEG drastically reduced erythrocytes toxic-ty and improved biocompatibility. The degree of functionalizationonsiderably reduced hemolysis by nearly 50% in case of DTX/FA-EG-MWCNTs, possibly due to the enhanced aqueous solubility andeparation of impurities.

MTT assay is a simple, non-radioactive, colorimetry based assayor determining the relative percent cell viability. The cytotoxic-ty of DTX loaded nanotubes formulations at different micromolaroncentration against MCF-7 cells after 24 and 48 h was deter-ined using MTT cytotoxicity assay. MTT assay revealed that upon

ncreasing the concentration from 0.001 to 100 �M of DTX loaded

anotubes formulations the relative percent cell viability of theancerous cells was decreased following initial 24 h treatment.fter 48 h, DTX exerted higher cytotoxicity as compared to 24 h

reatment due to sustained release of drug from the nanotubes

ig. 4. Percent cell viability of MCF-7 cells after treatment with free DTX, DTX/MWCNTs an = 3).

resented as mean ± S.D. (n = 3) “−, +, ++ and +++” indicate no change, small change,

formulations. The DTX/FA-PEG-MWCNTs exerted higher cytotox-icity as compared to DTX/MWCNTs and DTX solution and theincreased cytotoxic response was found to be concentration andexposure duration dependent. Folate receptors (FRs) are commontumor marker highly over-expressed on the cancerous cells surfacethat facilitates cellular internalization (Fig. 4). Thus, DTX/FA-PEG-MWCNTs formulation could efficiently deliver DTX to the nucleus ofthe cell possibly by nanoneedle-transporter or receptor-mediatedendocytosis (RME) mechanism [9,11,13,33].

We examined the effects of the developed nanotubes formula-tions on cell cycle in MCF-7 cells through flow cytometry. Generally,

cell cycle analysis could be characterized by the four distinct phasesin proliferating cell population: G1-, S-(DNA synthesis phase), G2-and M-phase (mitosis), while G2- and M-phases have an iden-tical DNA content and could not be discriminated on the basis

nd DTX/FA-PEG-MWCNTs at (A) 24, and (B) 48 h. Values represented as mean ± SD

N.K. Mehra, N.K. Jain / Colloids and Surfaces B: Biointerfaces 132 (2015) 17–26 23

F e (A)

M

osta8tt(ilaGgp

mttti

fgCcabTnFr

ig. 5. DNA content and cell cycle analysis (above) and quantitative cell uptake of thCF-7 cell lines using flow cytometry. Values represented as mean ± SD (n = 3).

f the DNA content [13,21,34]. The DNA flow cytometric analy-is (Fig. 5A–D) indicated that the treatment of MCF-7 cells withhe nanotubes formulations in 10 nM concentration caused 24 hrrest in G2 phase of the cell cycle. The control cells showed6.50 ± 3.22%, 8.15 ± 0.16% and 5.35 ± 0.08% population arrest inhe G1, G2 and S-phase, respectively. Percentage of cell arrest inhe G2 phase was found to be 42.29 ± 2.12% (DTX), 56.22 ± 1.56%DTX/MWCNTs), and 60.67 ± 2.02% (DTX/FA-PEG-MWCNTs) lead-ng to mitotic arrest in G2/M phase of the cell cycle that ultimatelyeads to cell death. The DTX/FA-PEG-MWCNTs cells arrest waspproximately 30.70 ± 1.24%, 60.67 ± 2.76% and 8.64 ± 0.22% in G1,2 and S-phase, respectively. Thus the DNA cell cycle analysis sug-ests that the cancerous cells were arrested significantly in the G2hase when treated with the DTX loaded formulation.

The DTX destroys cell’s ability to use its cytoskeleton in a flexibleanner binding with �-subunit of tubulin. DTX acts by binding

o microtubules and inhibits microtubule depolymerization to freeubulin. It has been reported that the nanotube formulation belongso cell-cycle specific anticancer drug, which mainly arrest the cellsn G2 phase of the cell cycle [14].

The quantitative cell uptake studies of developed nanotubeormulations were performed using flow cytometry to investi-ate the cellular uptake in MCF-7 cells. Fluorescence Activatedell Sorting (FACS) is a special type of flow cytometry, whichan quantitatively measure the cell uptake. A flow cytometernalyses particles by passing them in single file through a laseream and counts upto 1000 cells/s of fluorescence intensities.

he quantitative cellular uptake of the DTX from the developedanotubes formulations in MCF-7 cell is shown in Fig. 5A–D. InACS chromatograms, control group showed 69.16 ± 2.32% fluo-escence intensity in R1 region. The observed percent fluorescent

control, (B) DTX, (C) DTX/MWCNTs and (D) DTX/FA-PEG-MWCNTs formulations on

intensity of the DTX, DTX/MWCNTs and DTX/FA-PEG-MWCNTs wasfound to be 62.10 ± 3.04, 66.10 ± 3.22 and 77.72 ± 2.88%, respec-tively shifted toward R2 region. The observed higher fluorescenceintensity clearly suggests higher uptake of the DTX/FA-PEG-MWCNTs formulation.

The FA-terminated poly(ethylene glycol) (PEG-FA) coated onSWCNTs (DOX/PEG-FA/SWCNTs) in a facile non-covalent methodwas designed and constructed for targeting delivery of DOX tocancer cells. The DOX/PEG-FA/SWCNTs exhibit excellent stabilityunder neutral pH condition and selectively attach onto cancer cellsand enter the lysosomes or endosome by clathrin-mediated endo-cytosis [27]. Receptor-mediated cellular trafficking can facilitatecellular internalization of the drug loaded MWCNTs after conju-gation with targeting moiety. The in vitro release of FITC from theMWCNTs formulations showed negligible release (<1%) in 3 h, sug-gesting that only nanotubes formulations are internalized into theMCF-7 cell lines. FITC dye was covalently attached to nanotubes andalso on to interior wall of the nanotubes [5]. The obtained fluores-cence indicates the rapid internalization of nanotube formulations.The free FITC was washed away from the FITC loaded nanotube for-mulations prior to cellular uptake study. The FA-targeted MWCNTsmay increase the therapeutic index in a greater affinity for colorec-tal cancer cells than un-targeted MWCNTs [29]. Recently, Arora andco-workers reported the translocation and toxicity of the docetaxel(DTX) conjugated MWCNTs employing MCF-7 and MDA-MB-231human breast cancer cells. The DTX-MWCNTs conjugates indicateincreased efficacy over the drug in terms of cytotoxicity and thereby

enriching cancer therapies [35].

The pharmacokinetic study was performed to assess the effectof surface engineering on different pharmacokinetic parameterslike half value duration (HVD), area under the curve (AUC), area

24 N.K. Mehra, N.K. Jain / Colloids and Surfaces B: Biointerfaces 132 (2015) 17–26

Table 3Pharmacokinetic parameters of free DTX and DTX loaded MWCNTs formulations.

Parameters HVD (h) AUC(0–t)

(�g/mL h)AUC(0–∞)

(�g/mL h)AUMC(0–t)

(�g/mL h2)AUMC(0-∞)

(�g/mL h2)t1/2 (h) MRT (h)

Free DTX 0.34 ± 0.03 10.56 ± 0.65 11.02 ± 0.10 30.70 ± 0.46 38.15 ± 1.28 2.70 ± 0.02 3.45 ± 0.01DTX/MWCNTs 0.90 ± 0.05 22.17 ± 0.10 22.77 ± 0.18 127.24 ± 2.24 145.75 ± 4.55 4.64 ± 0.04 6.40 ± 0.02DTX/FA-PEG-MWCNTs 1.10 ± 0.08 33.67 ± 0.16 34.95 ± 0.34 328.35 ± 4.50 405.56 ± 6.54 8.81 ± 0.03 11.60 ± 0.07

MA 1/2 = elc

ulmD1taDttfdbpPsbts

tsttfip227w2MbD(

ean ± S.D. (n = 3); probability p < 0.001; standard deviation <5%.bbreviations: Cmax = peak plasma concentration; Tmax = time taken to reach Cmax; toncentration over time curve; HVD: half value duration.

nder the first moment plasma concentration curve (AUMC), half-ife (t1/2), and mean residence time (MRT) (Table 3 and Fig. 6). The

ean residence time (MRT) and t1/2 of free DTX, DTX/MWCNTs andTX/FA-PEG-MWCNTs were found to be 3.46 ± 0.01, 6.40 ± 0.02,1.60 ± 0.07 h and 2.70 ± 0.02, 4.64 ± 0.04, 8.81 ± 0.03 h, respec-ively. The MRT of DTX/FA-PEG-MWCNTs was found to bepproximately 3.5 and 1.85 folds higher compared to free DTX andTX/MWCNTs, respectively. The obtained results are attributed to

he biocompatibility of engineered nanotubes upon surface func-ionalization (degree of functionalization) and ability to resideor longer time inside the body. The improved pharmacokineticata make nanotubes as most promising, smart, and ideal nano-iocarrier for site-specific targeting. The sustained drug releaseatterns in blood were achieved to a greater extent for DTX/FA-EG-PMWCNTs as against DTX/MWCNTs and free DTX. It clearlyuggests the improved pharmacokinetic parameters with betterioavailability and prolonged retention in systemic circulation thanhat resulting from administration of drugs-MWCNTs and free drugolution to mice.

A comparative biodistribution study was performed to assesshe amount of drug that reaches in to different organs like liver,pleen, kidney, lungs and tumor after intravenous (i.v.) adminis-ration of free DTX, DTX/MWCNTs and DTX/FA-PEG-MWCNTs intohe tumor bearing Balb/c mice. In case of DTX/FA-PEG-MWCNTsormulation, higher concentration of DTX uptake was observedn tumor in 24 h. The DTX concentrations (percent injected doseer organ) from the DTX/MWCNTs determined at 1, 6, 12, and4 h were found to be 31.57 ± 0.18, 30.24 ± 0.17, 26.44 ± 0.15 and0.44 ± 0.88 in liver and 3.88 ± 0.06, 5.56 ± 0.86, 7.47 ± 0.65, and.67 ± 0.86 in tumor, respectively. The DTX concentrations in liverere found to be 38.67 ± 0.06, 36.65 ± 0.94, 32.34 ± 0.74, and

8.54 ± 0.32, respectively at 1, 6, 12 and 24 h from DTX/FA-PEG-

WCNTs. The DTX concentrations in tumor were determined to

e 9.45 ± 0.24, 12.56 ± 0.63, 13.01 ± 0.24, and 17.65 ± 0.18 fromTX/FA-PEG-MWCNTs, respectively at 1, 6, 12 and 24 h time points

Fig. 7).

Fig. 6. Plasma profile of free DTX and various nanotubes fo

imination half life; MRT = mean residence time; AUC(0–∞) area under plasma drug

The higher levels of the surface engineered MWCNTs observedat initial time point of administered dose in kidney and the rapiddecline in the overall formulation thereafter suggest that most ofthe nanotubes were eliminated through the renal excretion route.Researchers have reported no signs of toxicity due to accumulationin body/organs suggesting the utility of such systems in thera-peutic delivery [12]. In vitro drug release data suggested initialrapid release followed by gradual slow release; similar pattern wasobserved in in vivo study. The obtained data from the DTX/FA-PEG-MWCNTs formulations are in good agreement with the previouslypublished reports [9,13,26,29].

The in vivo tumor targeting efficacy of the DTX/MWCNTs andDTX/FA-PEG-MWCNTs was assayed on breast tumor model. Thestarting tumor size was 100 mm3 for all dose receiving groupsincluding developed nanoconjugates as well as normal salineand control group. The size of the tumor volume (mm3) at 30days after treatment of DTX/FA-PEG-MWCNTs was calculated tobe 57.0 ± 3.56. The reduced size of the tumor clearly suggeststhe better and efficient targeting of the developed nanotubeformulations. The DTX/FA-PEG-MWCNTs (targeted, stealth, longcirculatory nature) was found to be more active than DTX/MWCNTsand free DTX solution with significant reduction in tumor growth.The DTX loaded nanotubes formulations could be ranked in thefollowing order:

(DTX/FA-PEG-MWCNTs > DTX/MWCNTs > free DTX)(Maximum inhibitory Minimum inhibitory)

The higher antitumor activity of the targeted stealth nanotubeformulations could be ascribed to higher accumulation in cancer-ous cells via receptor-mediated endocytosis (R-ME) and passivediffusion (tiny nanoneedle) mechanism. However, FA appendedstealth nanotubes formulations were found to be significantly

more efficient in tumor suppression compared with plain MWC-NTs (non-targeted) and drug solution owing to the accelerated drugrelease from endosomes after internalization. Significant reductionin subsequent growth in tumor was probably due to ligand-driven

rmulations. Values represented as mean ± SD (n = 3).

N.K. Mehra, N.K. Jain / Colloids and Surfaces B: Biointerfaces 132 (2015) 17–26 25

Fig. 7. Biodistribution of DTX after intravenous administration of DTX solution, DTX/MWCN**p ≤ 0.01; ***p ≤ 0.001. ns: not significant vs. Free DTX. (Values represented as means ± S

Fr

igrctnN

be 1.02 ± 0.38, 7.71 ± 0.6 and 1.31 ± 0.12 × 103/�L, respectively.

TS

Vh

ig. 8. Kaplan–Meier survival curves of MCF-7 bearing Balb/c mice analyzed by Log-ank (Mental-Cox) test with normal saline group as control.

nternalization of the surface engineered nanotubes at the tar-et site/tissue(s), which was accompanied by slow and sustainedelease of the drug. The tumor growth inhibition study clearly indi-ates that inclusion of the pH-responsive characteristics increases

he overall targeting efficiency of the targeted and non-targetedanotubes formulations. The prepared surface engineered MWC-Ts formulations did not elicit any change in body weight of mice.

able 4erum biochemical parameters of Balb/c mice treated with free DTX, MWCNTs, DTX/MW

Group RBCs (×106/�L) WBCs (×106/�L) Differential

Monocytes

Control 9.21 ± 0.40 10.80 ± 0.40 1.40 ± 0.60Normal saline 8.40 ± 0.32 9.63 ± 0.42 0.91 ± 0.34Free DTX 5.82 ± 0.22 10.05 ± 0.32 0.92 ± 0.55MWCNTs 7.50 ± 0.56 8.60 ± 0.62 0.71 ± 0.76DTX/MWCNTs 8.02 ± 0.36 9.06 ± 0.62 0.90 ± 0.12DTX/FA-PEG-MWCNTs 8.81 ± 0.88 10.20 ± 0.60 1.02 ± 0.38

alues are expressed as mean ± SD. Number of animals per time points were three (n = 3)aematocrit.

Ts and DTX/FA-PEG-MWCNTs formulation in tumor bearing Balb/c mice). *p ≤ 0.05;D; n = 3).

The DTX loaded NGR peptide conjugated SWCNTs (DTX-NGR–SWCNTs) enhanced the targeting efficiency compared withDTX loaded SWCNTs (SWCNTs-DTX) and DTX alone [32].

Kaplan–Meier survival curves based on survival time were plot-ted for different groups of animals using Log-rank test. The curvessuggested that the tumor bearing mice of DTX/FA-PEG-MWCNTsexhibited significantly longer median survival time span (40 days,p < 0.001) than DTX/MWCNTs (24 days), free DTX (19 days) and con-trol group (12 days) (Fig. 8). These results further confirmed thehigher tumor treatment potential possessed by the surface engi-neered MWCNTs, which resulted in longer survival span of tumorbearing mice. The longest survival span was observed in case ofDTX/FA-PEG-MWCNTs.

Hematological parameters (RBCs, WBCs and differential counts)were determined to assess the relative effect of MWCNTs formu-lations (DTX/MWCNTs, and DTX/FA-PEG-MWCNTs) compared tofree DTX on different components of blood. The RBCs and WBCscounts in free DTX, MWCNTs, DTX/MWCNTs and DTX/FA-PEG-MWCNTs formulations treated blood sample were found to be5.82 ± 0.22, 7.50 ± 0.56, 8.02 ± 0.36, 8.81 ± 0.88 and 10.05 ± 0.32,8.60 ± 0.62, 9.06 ± 0.62, and 10.20 ± 0.60, respectively as shown inTable 4. The differential counts i.e. monocytes, lymphocytes andneutrophiles of DTX/FA-PEG-MWCNTs formulation were found to

The DOX/FA/CHI/SWCNTs did not exhibit obvious liver toxicity byblood routine and serum biochemical parameters on female nudeBalb/c mice [4]. The extensive data from the serum biochemical

CNTs and DTX/FA-PEG-MWCNTs formulations after 7 days.

counts (×103/�L) Hb (g/dL) HCT

Lymphocytes Neutrophils

7.92 ± 0.42 1.62 ± 0.42 12.40 ± 0.33 35.50 ± 0.65 6.11 ± 0.44 1.00 ± 0.66 10.50 ± 0.22 34.40 ± 0.25 7.93 ± 0.12 1.41 ± 0.60 10.22 ± 0.15 32.40 ± 0.16 5.91 ± 0.88 0.90 ± 0.85 9.80 ± 0.94 33.62 ± 0.12 6.82 ± 0.90 1.01 ± 0.40 10.81 ± 0.22 33.82 ± 0.45 7.71 ± 0.60 1.31 ± 0.12 11.80 ± .90 34.02 ± 0.12

; WBCs: white blood corpuscles, RBCs: red blood corpuscles, Hb; hemoglobin, HCT;

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4

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A

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6 N.K. Mehra, N.K. Jain / Colloids and S

arameters suggest that the RBCs, WBCs and differential count ofhe DTX/FA-PEG-MWCNTs were almost similar to the control andormal saline treated groups. Similarly, the differential counts i.e.

eucocytes, monocytes and lymphocytes were found almost similarn case of DTX/FA-PEG-MWCNTs nanoconjugates to normal values.he results clearly establish the superior biocompatibility of theolate appended MWCNTs than the pristine MWCNTs and free DTXolution.

. Conclusion

The highly effective novel targeted drug delivery system basedn FA conjugated and PEGylated MWCNTs was developed andvaluated in facile strategy for cancer treatment. The DTX/FA-PEG-WCNTs formulation showed higher cytotoxicity as compared toTX/MWCNTs and free drug solution and arrested cell death in G2hase. In contrast, quantitative cell uptake demonstrated signifi-antly higher uptake of FA conjugated nanotubes formulations. Thex vivo studies such as MTT cytotoxicity, DNA cell cycle, and quan-itative cell uptake clearly revealed that the targeted drug deliverylong with specific targeting moiety increases the receptor interac-ion for selective killing of MCF-7 cells. The pharmacokinetic studieslso revealed the long circulatory (stealth) nature of the devel-ped MWCNTs formulations. In vivo toxicity studies suggest thathe surface engineered MWCNTs formulations easily escaped fromhe excretory organ. The degree of functionalization minimizes theoxicity of the carbon nanotubes. The developed surface engineered

WCNTs nanoconjugates have shown promising potential in can-er therapy and to deliver significantly higher concentration of DTXo the cancerous tissue than pristine MWCNTs and free drug. Thus,t may be concluded that the DTX laden FA-PEG-MWCNTs holdstrong targeting potential in cancer treatment.

eclaration of interest

The authors report no conflict of interest.

cknowledgement

One of the author Dr. Neelesh Kumar Mehra is thankful to theniversity Grants Commission (UGC), New Delhi, India for provid-

ng the Senior Research Fellowship during the tenure of the studies.he authors also acknowledge Dr. Ranveer Kumar, Department ofhysics, Dr. H. S. Gour University, Sagar, India for Raman spec-roscopy; Central Instruments Facilities (CIF), National Institute of

harmaceutical Education and Research (NIPER), Mohali, Chandi-arh, India for Particle Size analysis; Central Drug Research InstituteCDRI), Lucknow, India for FTIR spectroscopy; All India Institutef Medicine and Sciences (AIIMS), New Delhi, India for electron

[

[[

s B: Biointerfaces 132 (2015) 17–26

microscopy; Indian Institute of Technology (IIT), Indore, India forAFM analysis; Diya Laboratory, Mumbai, India for XRD analysis andNational Center for Cell Sciences (NCCS), Pune, India for providingthe cell line.

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