19 Mehra et al Biomaterials 2014 Research

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The cancer targeting potential of D-a-tocopheryl polyethylene glycol1000 succinate tethered multi walled carbon nanotubes

Neelesh Kumar Mehra a, Ashwni Kumar Verma b, P.R. Mishra b, N.K. Jain a,*

a Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour Central University, Sagar 470 003, Indiab Pharmaceutics Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, Uttar Pradesh, India

a r t i c l e i n f o

Article history:Received 19 December 2013Accepted 12 February 2014Available online 4 March 2014

Keywords:Carbon nanotubesDoxorubicin hydrochlorideVitamin EKaplaneMeier survivalTumor growth inhibitionAnticancer activity

a b s t r a c t

Our main aim in the present investigation was to explore the in vitro and in vivo cancer targeting po-tential of the doxorubicin (DOX) laden D-a-tocopheryl polyethylene glycol 1000 succinate (vitamin ETPGS) tethered surface engineered MWCNTs nanoformulation (DOX/TPGS-MWCNTs) and compare itwith pristine MWCNTs and free doxorubicin solution. The developed MWCNTs nanoformulations wereextensively characterized by Fourier-transform infrared, Raman spectroscopy, x-ray diffraction, electronmicroscopy, and in vitro and in vivo studies using MCF-7 cancer cell line. The entrapment efficiency wasdetermined to be 97.2 � 2.50% (DOX/TPGS-MWCNTs) and 92.5 � 2.62% (DOX/MWCNTs) ascribed to p-pstacking interactions. The developed formulations depicted the sustained release pattern at the lyso-somal pH (pH 5.3). The DOX/TPGS-MWCNTs showed enhanced cytotoxicity, cellular uptake and weremost preferentially taken up by the cancerous cells via endocytosis mechanism. The DOX/TPGS-MWCNTsnanoconjugate depicted the significantly longer survival span (44 days, p < 0.001) than DOX/MWCNTs(23 days), free DOX (18 days) and control group (12 days). The obtained results also support the extendedresidence time and sustained release profile of the drug loaded surface engineered nanotubes formu-lations in body as compared to DOX solution. Overall we can conclude that the developed MWCNTsnanoconjugate have higher cancer targeting potential on tumor bearing Balb/c mice.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Currently, surface engineeredmultifunctional carbon nanotubes(CNTs) mediated targeted and controlled drug delivery has arousedescalating attention as valuable, promising nano-architecture dueto its unique physicochemical properties in the treatment of cancerand other dreadly diseases [1e4]. CNTs was originally discoveredand fully described by Prof. Sumio Iijima in 1991 and it is consid-ered as promising targeted drug delivery vehicles because it caneasily cross cell membranes. CNTs is three dimensional, cylindrical,sp2 hybridized carbon nanomaterial. CNTs can be subdivided intosingle-, double-, triple-, and multi-walled CNTs [2,5e7].

The hydrophobic nature and inherent toxicity of first generationpristine CNTs make them unsuitable for targeted/controlled drugdelivery. However, these major hurdles have been easily amelio-rated by surface alteration through either covalent or non-covalent

approaches depending on the intermolecular interaction. The non-covalent alteration is based on the extended p-system (p-orbital) ofthe sidewall of the nanotubes bind with the guest moieties throughpep stacking interactions [2,6,8,9]. The surface engineered CNTsserves as efficient multifunctional biological transporters devoid ofobvious toxicities. Iverson et al. reported that the alginate-encapsulated single-walled carbon nanotubes (SWCNTs) did notshow any adverse response for more than 400 days [10]. Thus,surface engineered CNTs has been designed and tested for targeteddelivery by conjugating targeting moieties and has proven non-cytotoxic to human cells [11,12].

Doxorubicin (DOX), an anthracycline antibiotic, is a DNA-interacting drug for treatment of various cancers especiallybreast, ovarian, prostate, brain, cervix and lung cancers. Clinicalapplication of doxorubicin is limited because of its short half-lifeand severe toxicity to normal tissues, especially gastrointestinaltoxicity and heart failure. The cardio-toxicity confines the cumu-lative dose of DOX to 500e600 mg/m2, which still can be increasedfor tumor but not for heart disease [13e15]. Our group is continu-ously working and exploring the drug delivery aspects employingthe surface engineered CNTs for targeting purpose including

* Corresponding author. Tel./fax: þ91 7582 265055.E-mail addresses: [email protected] (N.K. Mehra), [email protected]

(N.K. Jain).

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Biomaterials 35 (2014) 4573e4588


Doxorubicin [9,15,16], Gemcitabine [17], Sulfasalazine [5], andAmphotericin B [12]. Recently, our group reported the targeteddelivery of DOX using folic acid conjugated PEGylated MWCNTswith improved therapeutic outcomes [14].

D-a-tocopheryl polyethylene glycol 1000 succinate (vitamin ETPGS; TPGS) is FDA approved water-soluble derivative of naturalVitamin E (PEGylated vitamin E). TPGS is prepared by esterificationof D-a-tocopheryl acid succinate and PEG 1000 (amphiphilicvitamin E) and moderately stable under normal conditions[13,18,19]. TPGS is a promising surfactant for green processing ofthe carbon based nanomaterials including CNTs and offers as analternative and valuable option enhancing the aqueous solubility,avoiding multidrug resistance (MDR) and might elicit receptor-mediated endocytosis (RME). Currently, TPGS is most widely usedto enhance the cellular uptake, cytotoxicity of the anticancer agentslike Doxorubicin, Paclitaxel and Vinblastine etc. The TPGS-basedconjugates are ideal solution for the bioactive(s), which haveobstacle in ADME characteristics [13].

In the present investigation, we surface engineered and deco-rated MWCNTs with the targeting moiety TPGS laden with doxo-rubicin (DOX/TPGS-MWCNTs) assessing the cancer targetingpotential and compared to free DOX solution in the tumor bearingBalb/c mice. We also determined the pharmacokinetics, bio-distribution, Kaplan Meier survival analysis, tumor growth inhibi-tion study and toxicological aspects in terms of safety and efficacy.It is believed that the TPGS anchored surface engineered MWCNTsare adsorbed with apo-lipoproteins (ApoE), which interact withLDL receptors and are internalized via RME mechanism [20,21].

2. Materials and methods

The pristine multi walled carbon nanotubes (MWCNTs) produced by CatalyticChemical Vapor Deposition (CCVD) with 99.3% purity, were purchased from SigmaAldrich Pvt. Ltd. (St. Louis, Missouri, USA) was used for the present studies. Cancercell lines were purchased from the National Centre for Cell Sciences (NCCS) Pune,India. Poly-tetrafluoroethylene (PTFE) filters (0.22 mm pore size) were purchasedfrom Hangzhou Anow Microfiltration Co. Ltd., Hangzhou, China. Sulfuric acid, nitricacid, thionyl chloride, ethylene diamine, succinic anhydride, dimethyl amino pyri-dine, dichloromethane, dimethyl sulfoxide (DMSO), and 1-Ethyl-3-(3-dimethylaminopro-pyl) carbodiimide (EDC) were purchased from HiMedia Pvt.Ltd. Mumbai, India. All the reagents and solvents were used as received.

2.1. Purification and surface engineering of pristine MWCNTs

As-procured pristine MWCNTs were initially purified using vacuum oven andmicrowave technique as previously reported by Mehra and Jain [14]. PurifiedMWCNTs were then further used for functionalization in the subsequent steps likecarboxylation, acylation and amidation process, extensively characterized and re-ported [5,9,12,14,16,17,22].

2.2. Conjugation of TPGS to surface engineered MWCNTs

2.2.1. Synthesis of succinoylated TPGSThe carboxylic derivative of TPGS (TPGS-COOH) was activated by succinic an-

hydride (SA) through ring-opening reaction in the presence of 4-dimethylaminopyridine (DMAP) with slight modification of previously reportedmethod [13]. Briefly, TPGS (0.77 g, 0.5 mM), SA (0.10 g,1mM) and DMAP (0.12 g,1mM)were mixed and heated at 100 � 5 �C under nitrogen gas protection at room tem-perature (RT) for 24 h; themixturewas cooled to room temperature (RT), taken up in5.0 mL cold dichloromethane (DCM), filtered to remove excess SA and precipitatedin 100 mL diethyl ether at �10 �C overnight. The obtained white precipitant wasfiltered and dried in vacuum to obtain succinoylated TPGS (Scheme 1).

2.2.2. Conjugation of TPGSeCOOH to amine terminated MWCNTsThe TPGSeCOOH was reacted with amine terminated MWCNTs (MWCNTse

NH2) using EDC chemistry with slight modifications [14,23]. The amine terminatedMWCNTs and TPGSeCOOH were reacted in DMSO at 1:2 ratio (excess amount ofTPGSeCOOH) with continuous magnetic stirring for 2 days at room temperature.The unconjugated TPGS-COOH was removed by dialysis method and MWCNTsconjugate was collected, freeze dried and characterized (Scheme 2).

2.3. Drug loading

Briefly, DOX was dissolved in acetone (10 mg/mL), and approximately 1.2 mLaqueous triethyl amine (TEA) solution was added in a molar ratio of 2:1 (DOX:TEA).

The solution was magnetically stirred overnight using Teflon bead and mixed withthe dispersion of MWCNTs in PBS (pH 7.4) with the same ionic strength adjusted byaddition of sodium chloride (NaCl). The DOX:MWCNTsmixture in optimized 1:2 (w/w) ratio was magnetically stirred overnight (50 rpm; Remi, India) at 37 � 0.5 �C for24 h using Teflon bead to facilitate entrapment of DOX. Thereafter, DOX ladenMWCNTs were separated by the centrifugation to remove free/unbound DOX untilsolution became color free, and measured at lmax 480.2 nm spectrophotometrically(Shimadzu 1601, UV-Visible Spectrophotometer, Shimadzu, Japan) using a calibra-tion curve prepared under the same condition [14,24]. The DOX loading efficiencywas calculated spectrophotometrically using the following formula:

% Loading Efficiency ¼ Weight of loaded DOX�Weight of free DOXWeight of loaded DOX

� 100

The product was collected, dried and lyophilized (Heto dry winner, Denmark,Germany) and stored at 5 � 3 �C for further use of studies.

2.4. Characterization of pristine and engineered MWCNTs

The pristine MWCNTs (as procured) and TPGS functionalized MWCNTs werecharacterized using Fourier Transform Infrared (FTIR), Raman, x-ray diffraction(XRD), particle size and particle size distribution measurement.

2.4.1. FTIR spectroscopyThe FTIR spectroscopy were performed using compressed KBr pellet method in

Perkin Elmer FTIR spectrophotometer (Perkin Elmer 783, Pyrogon 1000 Spectro-photometer, Shelton, Connecticut) and scanned in the range from 4000 to 500 cm�1.

2.4.2. Average particle size and particle size distribution (PSD) measurementThe average particle size and particle size distribution (PSD) of pristine and

surface engineered MWCNTs were determined by photon correlation spectroscopyin a Malvern Zetasizer nano ZS90 (Malvern Instruments, Ltd, Malvern, UK) at roomtemperature (RT).

2.4.3. Electron microscopyThe size and surface morphology were characterized by Transmission Electron

Microscopy (TEM; Morgagni 268-D, Fei Electron Optics, Holland) after drying oncarbon-coated copper grid and staining negatively by 1% phosphotungstic acid (PTA)by metal shadowing technique.

2.4.4. X-ray diffraction (XRD) analysisThe X-ray diffraction (XRD) analysis of the pristine and surface engineered

MWCNTs was carried out using X-ray diffractometer (PW 1710 Rigaku, San Jose, CA)

Scheme 1. Schematic representation of the activation of the TPGS (TPGSeCOOH).

N.K. Mehra et al. / Biomaterials 35 (2014) 4573e45884574

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by adjusting X-ray power of 40 kV and 40 mA. Three hours of exposure time wastaken to analyze samples and X-ray diffraction data was obtained by general areadetector diffraction system at 25 �C.

2.4.5. Raman spectroscopyThe order-disorder hexagonal carbon and the Raman spectra (Strokes lines)

were recorded by a Raman micro-spectroscopy RINSHAW, inVia Raman Spec-trophotometer (Renishaw, Gloucestershire, UK). The micro-spectrophotometerwas operated exciting with the 532 nm laser radiation under objective lens of20� magnification (Olympus BX 41, USA) with a slit of 1 � 6 mm and an incidentpower was around 1 mW. The exposure time was 30 s and three scans wereaccumulated for each spectrum. All the spectra were recorded at 0.1 cm�1 stepintervals at RT. Additionally, to protect from damage by the laser beam, thesample was embedded into a KBr pellet and low power of 1.2 mW was employedon the surface of sample to minimize appreciable peak shift or peak broadeningcaused by the laser heating.

2.5. In vitro release studies

The in vitro release of DOX from DOX/MWCNTs and DOX/TPGS-MWCNTsnanoformulation was studied in sodium acetate buffer saline (pH 5.3) and phos-phate buffer saline (pH 7.4) as recipient media in a modified dissolution method

maintaining the physiological temperature (37 � 0.5 �C) throughout the study [14].The dialysis membrane (MWCO 5e6 kDa, HiMedia, India) filled with the developedoptimized nanotubes formulations separately, hermetically tied at both ends andimmediately placed into the receptor media maintaining strict sink conditions withconstant stirring using magnetic stirrer at RT adjusted to 37 � 0.5 �C (100 RPM;Remi, Mumbai, India). The aliquots were withdrawn at different time points andvolume of recipient compartment was maintained by replenishing with fresh sinksolution. The DOX concentration was determined in triplicate at different timepoints after appropriate dilutions by UV/Visible spectrophotometer at lmax 480.2 nm(UV/Vis, Shimadzu 1601, Kyoto, Japan).

2.6. Stability study

The DOX laden optimized developed MWCNTs nanoformulations (DOX/MWCNTs and DOX/TPGS-MWCNTs) were stored in dark and in amber colored andcolorless glass vials at 5 � 3 �C, room temperature (25 � 2 �C) and at 40 � 2 �C for aperiod of six months in stability chambers (Remi CHM-6S, India) as per “Interna-tional Conference on Harmonization of Technical Requirements for Registration ofPharmaceuticals for Human Use” (ICH) guidelines for finished pharmaceutical drugproducts [25]. The MWCNTs nanoformulations were analyzed initially and period-ically upto six months for any change in particle size, drug content and organolepticfeatures like aggregation and precipitation, color and odor changes if any.

Scheme 2. Schematic representation of the TPGS functionalized MWCNTs.

N.K. Mehra et al. / Biomaterials 35 (2014) 4573e4588 4575


2.7. Comparison of hemolytic toxicity study

The erythrocytes-nanotubes formulations interaction was performed in vitrofollowing previously reported procedure, with minor modification [14,17]. Briefly,fresh whole human blood was collected in HiAnticlot blood collecting vials(HiMedia, Mumbai, India) and centrifuged at 3000 rpm (Remi, Mumbai, India) for15 min in an ultracentrifuge (Z36HK, HERMLE LaborTchnik GmbH, Germany). Thered blood corpuscles (RBCs) were collected from the bottom and separated out,washed with physiological normal saline (0.9%; w/v) until clear, colorless superna-tant was obtained above the cell mass. The RBCs suspension (1 mL) was mixed withthe 0.9% w/v normal saline (4.5 mL), free DOX, DOX/MWCNTs and DOX/TPGS-MWCNTs dispersions (0.5 mL) incubated for 60 min; and allowed to interact. Afterincubation, samples were centrifuged for 15 min at 1500 rpm and supernatant wastaken to quantify the hemoglobin content at lmax 540 nm spectrophotometricallyconsidering 0.9% w/v NaCl solution (normal saline) and deionized water as nil and100% hemolysis, respectively. The percent hemolysis was calculated using theformula.

Hemolysis % ¼ ðAbs� Abs0ÞðAbs100 � Abs0Þ

� 100

where, Abs, Abs0 and Abs100 represent the absorbance of samples, a solution of 0%hemolysis and a solution of 100% hemolysis, respectively.

2.8. Cell line studies

The MCF-7 (Michigan Cancer Foundation-7; an estrogen receptor positive hu-man breast cancer cell line derived from pleural effusion) cell line was procuredfrom National Center for Cell Sciences (NCCS), Pune, India for the present study. TheMCF-7 cells were cultured in a humidified atmosphere containing atmosphere at 5%CO2 at 37 �C in Dulbecco’s Modified Eagle Medium (DMEM; HiMedia, Mumbai, In-dia) containing 10% fetal bovine serum (FBS; HiMedia, Mumbai, India) supple-mented with 2 mM L-glutamine, 1% penicillinestreptomycin mixture (Sigma, StLouis, Missouri) incubated for 24 h for more than 80% confluence. The mediumwaschanged two to three times in a week [14,26,27].

2.8.1. Methylthiazole tetrazolium (MTT) cytotoxicity assayThe methylthiazole tetrazolium (MTT) cytotoxicity assay was performed by

cleavage of tetrazolium salt [{3-(4,5 dimethyl thiazole-2 yl)-2,5-diphenyl tetrazo-lium bromide} (MTT)] to a blue formazan derivative by living cells [14,23,26,28].Briefly, MCF-7 cells were seeded in 96-well plates with density 1 �104 cells per welland allowed to adhere for 24 h at 37 �C prior to assay. Then the cells in quadrupletwells were treated with free DOX, DOX/MWCNTs, and DOX/TPGS-MWCNTs atconcentrations-0.01, 0.1, 10, 100 mM for 24 h. Thereafter, medium was decanted and50 mL of methylthiazole tetrazolium (MTT) (1 mg/mL) in DMEM ((10 mL; 5 mg/ml inHank’s Balanced Salt Solution; without phenol red) was added to each well andincubated at 37 �C for 4 h. MTT is reduced by mitochondrial dehydrogenase activityin metabolically active cells to form insoluble formazan crystals. The formazancrystals were solubilized in 50 mL isopropanol by shaking at room temperature for10 min. Absorbance was measured at 570 nm. The absorbance given by untreatedcells was taken as 100% cell survival and the relative (%) cell viability was calculatedusing following formula:

Cell viabilityð%Þ ¼ ½A�test½A�control

� 100

where, [A]test is the absorbance of the test sample and [A]control is the absorbance ofcontrol samples.

2.8.2. Cell cycle analysis and sub-G1 DNA measurementThe cultured MCF-7 cells were seeded in 1 � 104 cells per well in 6-well plates

and incubated for 24 h. MWCNTs formulations (2 nM/mL concentration) were addedinto each well and incubated for 24 h. After incubation the cells were harvested bycentrifugation at 1000 � g for 10 min, washed with ice-cold PBS and fixed using 70%cold ethanol overnight. The fixed cells were suspended in pre-cold PBS and furthertreated with RNase (DNase free, 100 mg/mL) and propidium iodide (PI; 50 mg/mL) for30 min at 37 �C in dark. The treated cells were centrifuged and obtained cell pelletswere re-suspended with PBS and kept on ice till used. The number of cells indifferent phases of the cell cycle was determined using the cell cycle analysis soft-ware with FACSCalibur Flow Cytometer (Becton, Dickinson Systems, FACS canto�,USA) [29].

2.8.3. Cell uptake/fluorescence microscope studiesThe qualitative and quantitative cellular uptake of the DOX from the DOX loaded

nanotubes formulation was performed using FACSCalibur Flow Cytometer (Becton,Dickinson Systems, FACS canto�, USA). The developed formulations and free drugsolution were incubated as in case of DNA cell cycle content, for 4 h, and then themedium was removed, the cells were washed with cold-PBS three times andanalyzed quantitatively (FACSCalibur Flow Cytometer (Becton, Dickinson Systems,FACS canto�, USA) and qualitatively (Inverted microscope; Leica, Germany)[13,21,23].

2.9. In vivo studies

The Balb/c mice of either sex (20e25 g) were used for present in vivo studies inaccordance with the guidelines by Committee for the Purpose of Control and Su-pervision of Experiments on Animals (CPCSEA) Registration No. 379/01/ab/CPCSEA/02 of Dr. H.S. Gour Vishwavidyalaya, Sagar, (M.P.). India. All the experimental animalprotocols were approved by the Institutional Animal Ethics Committee and animalswere acclimatized at room temperature by maintaining the relative humidity (RH)55e60% under natural light/dark condition prior to studies. The tumor model wasgenerated by injected serum-free MCF-7 cells (1 � 107 cells) using hypodermicneedle subcutaneously in the right hind leg of the mice and routinely monitored fortumor development by palpating the injected area with index finger and thumb forthe presence of the tumor (approximately 100 mm3) [14,30].

2.9.1. Analysis of pharmacokinetic parameter after intravenous (i.v.) administrationThe different pharmacokinetic parameters were determined after i.v. adminis-

tration of free DOX and DOX laden MWCNTs nanoformulations with the same i.v.dose (5.0 mg/kg body weight dose). The blood samples were collected from theretro-orbital plexus of eyes with mild anesthesia conditions into the Hi-Anticlotblood collecting vials (HiMedia, Mumbai, India) at predetermined time pointsupto 48 h. The collected of blood samples were centrifuged to separate RBCs andsupernatant (serum) was collected, 100 mL trichloro acetic acid (TCA) in methanol(10% w/v) was added, vortexed and ultracentrifuged (Z36HK, HERMLE LaborTchnikGmbH, Germany). The clear supernatant was collected and DOX concentration wasdetermined by High Performance Liquid Chromatography (HPLC) method anddifferent pharmacokinetic parameters were calculated [14,31e33].

2.9.2. Tissue/organ biodistribution studyThe in vivo biodistribution of the DOX ladenMWCNTs formulations and free DOX

were studied on tumor bearing Balb/c mice. The sterilized free DOX, DOX/MWCNTsand DOX/TPGS-MWCNTs conjugates after dispersion in normal saline (0.9%; w/v)were administered intravenously through caudal tail vein route (equivalent dose ofDOX ¼ 5.0 mg/kg body weight) into animals. Each mice was administered the samei.v. dose and carefully sacrificed by decapitation method at time intervals of 1, 6, 12and 24 h for the collection of visceral organs like liver, spleen, kidney, heart, andtumor immediately. The collected organs were washed with Ringer’s solution toseparate any adhered debris and dried with the help of tissue paper, weighed andstored under frozen till used. Tissues were homogenized (York Scientific Instrument,New Delhi, India) and vortexed after addition of chloroform (CHCl3) and methanol(CH3OH) mixture and ultracentrifuged at 3000 rpm for 15 min (Z36HK, HERMLELaborTchnik GmbH, Germany). After centrifugation, obtained supernatant wasdecanted into another vial and evaporated to dryness under nitrogen gas in a bath at60 � 2 �C temperature. The dried residue was collected in vials and injected in to anHPLC and analyzed for DOX content by HPLC (Shimadzu, C18, Japan) method,whereinmobile phase consisted of buffer pH 4.0/acetonitrile/methanol (60:24:16; v/v/v) with 1.2 mL/min flow rate at 102/101 bars pressure with adjusting 20 minruntime and peak at 480.2 nmwas considered with its retention time (RT) and area.

2.9.3. Assessment of anti-tumor cancer targeting efficacyThe in-vivo anti-tumor cancer targeting efficacy of the DOX laden MWCNTs

formulation was assessed in the tumor bearing Balb/c mice. The initial tumor sizewas taken approximately 100 mm3 in size. The tumor bearing mice were randomlydivided into four treatment groups (control, free DOX, DOX/MWCNTs and DOX/TPGS-MWCNTs) for treatment with 5.0 mg/kg body weight dose equivalent toDOX. At predetermined time intervals the tumor volume (cubic millimeters) wasmeasured by measuring its dimension (major and minor axis) using electronicdigital Vernier Caliper. The formula used to compute tumor volume was similar tovolume of an ellipsoid, where V¼ 4/3 p(1/2 length� 1/2 width� 1/2 depth) with anassumption that width is equal to depth and p equals 3, and so the final formulaused was V ¼ 1/2 � length � width2. The median survival time was also recorded.The study was terminated 45 days post treatment. All animals were accommodatedin a pathogen-free laboratory environment during the tenure of the studies.

2.9.4. Toxicological assessmentThe various toxicological aspects like hematological parameters and hepato-

toxicity were determined.

2.9.4.1. Hematological studies. The hematological parameters were estimatedfollowing the earlier reported method [5,14]. The mice were divided in respectivegroups and administered the same i.v. dose of the developed nanotubes formula-tions and free drug solution andmaintained on same regular diet upto 7 days. After 7days blood samples were collected from the mice and the red blood corpuscles(RBCs), white blood corpuscles (WBCs) and differential count of monocytes, lym-phocytes and neutrophils, % Hb, MCH and HCT were determined.

2.9.4.2. Hepatotoxicity. The serum enzyme activities such as creatinine levels,lactate dehydrogenase (LDH), blood urea nitrogen (BUN), SGPT (Serum glutamicpyruvic transaminase) were assayed using commercially available kit (Crest Bio-system, India) [26,27,34].



2.10. Statistical analysis

The results are expressed as mean � standard deviation (�SD) (n ¼ 3) andstatistical analysis was performed with Graph Pad Instat Software (Version 3.00,Graph Pad Software, San Diego, California, USA) by one-way ANOVA followed byTukeyeKramer test for multiple comparisons. The pharmacokinetic data analysis ofplasma concentration time profile was conducted using the Kinetica software(Thermo scientific), USA followed by non-compartment analysis. A probabilityp � 0.05 was considered while significant and p � 0.001 was considered asextremely significant.

3. Results and discussion

The surface engineered carbon nanotubes (CNTs) play a pivotalrole and present new opportunities for research and developmentincluding drug targeting arena. We procured pristine MWCNTs,purified using microwave oven (separate the impurities) and cut(shorten nanotubes) prior to use. The longer nanotubes are unableto enter most of the cancerous cells and may be toxic. Oxidation isone of the most common and prerequisite technique for intro-ducing the hydrophilic functional groups (carboxylic, phenolic andlactone etc) at the ends and side wall of the nanotubes throughstrong oxidizing acid treatment increasing aqueous dispersibility ofthe nanotubes [14,35,36].

In this purification, carboxyl (eCOOH) functional groups weregenerated onto the CNTs surface making them more safe withimproved aqueous solubility for precise and targeted drug delivery.We previously reported the purification, oxidation of pristine

MWCNTs and determined the total functional groups and carbox-ylic acid (eCOOH) by Boehm Titration method [14].

The TPGSeCOOH was conjugated to the amine terminatedMWCNTs through EDC chemistry. The FTIR spectra of TPGS, pristineMWCNTs and functionalized MWCNTs are shown in Fig. 1.

The FTIR spectrum of procured unmodified (pristine) MWCNTsdepicts absorption peak at 1626 cm�1, confirming the presence ofcarbon residue on the nanotubes surface. A clear single peak at2400.24 cm�1, which could be ascribed to the stretching of thecarbon nanotubes backbone is another important characteristic(Fig. 1 A).

The acid functionalized MWCNTs (MWCNTseCOOH) shows thepeaks at around 3425.6, 1637.4 and 1370.1 cm�1. The peak at1637.4 cm�1 is attributable to asymmetrical stretching of C]Ostretching vibration mode that was ascribed the expansion ofcarboxylation on the MWCNTs, and a peak at 3425.6 cm�1 wasascribed to the OeH stretching vibration (Fig. 1B). The blue shiftobserved in the carboxyl stretching may be a consequence ofintroduction of hydrogen bond amongst the surface carboxylicfunctional groups.

The FTIR spectrum of as-received TPGS shows characteristicpeak at 3088.17, 2876.31, 2788.22, 1682.21, 1513.21, 1428.12,1243.64, and 1182.93 cm�1. The aromatic stretching was found at3088.17 cm�1, while 2876.31 cm�1 and 2788.22 cm�1shows thealiphatic stretching for asymmetric and symmetric characteristicpeak (CeH stretching of the CH3). The C]O stretching shows peak

Fig. 1. FTIR spectra of (A) pristine MWCNTs, and (B) oxidized MWCNTs. FTIR spectra of (C) TPGS, and (D) TPGS-MWCNTs.



at 1682.21 cm�1, approximately. The C]C characteristic ringstretching was observed at 1513.21 and 1428.12 cm�1. The CeOstretching was observed at 1243.64 and 1182.93 cm�1. The obtainedcharacteristic peaks suggest that the TPGS is pure and authentic(Fig. 1C).

The FTIR spectrum of the TPGS conjugated MWCNTs showscharacteristic peaks at 1689.20 cm�1 of eC]O stretching ofamide bond formation, 2943.41 cm�1 due to CeH stretching ofCH2 functional group, and 3412.76 cm�1 of NeH stretching. Theobtained characteristic peaks of the as-received TPGS indicatethat the TPGS was successfully conjugated with the carboxylicgroup (-COOH) of the MWCNTs through amide bond formation(Fig. 1D).

The average particle size (nm) and particle size distributionwithpolydispersity index (PDI) were determined by photon correlationspectroscopy in a Malvern Zetasizer nano ZS90 (Malvern In-struments, Ltd, Malvern, UK) at room temperature (RT). The par-ticle size of the purified MWCNTs through microwave treatmentwas found to be 1254 � 5.88 nm with polydispersity index (PDI)0.429 � 0.23, however upon chemical treatment the size of thefunctionalized MWCNTs reduced. The average particle size andparticle size distribution of the DOX/MWCNTs and DOX/TPGS-MWCNTs were found to be 230.41 � 1.3 and 250.18 � 5.5 nmwith polydispersity index (PDI) of 0.27 � 0.010 and 0.32 � 0.008,respectively (Table 1). The average particle size and PDI clearlysuggest that the developed nanotubes formulation have narrow

Fig. 1. (continued).



particle size distribution with low polydispersity index (PDI). Renet al. reported the size & PDI of DOX loaded oxidized angiopep-2conjugated PEG-MWCNTs (DOX-O-MWCNTs-PEG-ANG) to be202.23 � 3.43 nm and 0.342 � 0.016, respectively [30].

Transmission electron microscopy (TEM) was used to investi-gate the possible morphological changes of MWCNTs depending onthe severity of each oxidizing treatment and find out any increasein size owing TPGS conjugation on functionalized MWCNTs. TheTEM photomicrographs of pristine, oxidized and TPGS tetheredMWCNTs are shown in Fig. 2. Results showed that the size wasreduced after oxidation and increase in time of oxidation,decreased the size of MWCNTs. The TEM photomicrographs of theTPGS-MWCNTs clearly revealed that the developed optimizednanotubes nanoformulation was in nanometric size range evenafter conjugation. The developed TPGS-MWCNTs conjugate showedgood dispersibility and stability at RT.

Raman spectroscopy technique is a very valuable and commonlyused spectroscopic method for characterization of CNTs. It providesthe information on the hybridization state and defect concentrationof the CNTs. It also gives the information about slight structuralchanges of MWCNTs, and changes in electronic structure of theattached functional moieties [22,37,38].

Carbon nanotubes usually have following four bands in Ramanspectra: (1) Radial breathing mode (RBM) in 100e400 cm�1 region,which is inversely proportional to the diameter of the nanotubes;(2) the disorder-related so-called D mode, approximately at 1330e1360 cm�1 provides information regarding amorphous impuritiesand carbon nanotubes wall disorders; (3) high-energy modeknown as tangential G band (HEM, often called G mode in the re-gion of 1500e1600 cm�1) caused by stretching along the CeCbonds in the graphitic plane; and (4) the D’mode at approximately1615 cm�1. The RBM mode is a characteristic for single walledcarbon nanotubes (SWCNTs) only that is caused by uni-axial vi-brations of CNTs. In most cases, MWCNTs do not show this signal,instead they show the D’band, which is assigned to the in-plane

vibrations of graphite [37e39]. The Raman spectra of the pristineand surface engineered MWCNTs are shown in Fig. 3. The Ramanspectrum of the pristine MWCNTs shows the Raman shift at1579.85 cm�1 and at 1346.15 cm�1, which correspond to the G band(graphite-like mode) and D band (disorder-induced band),respectively. As the MWCNTs were oxidized (carboxylated), G bandwas shifted to 1584.73 cm�1 and D band to 1352 cm�1. The Ramanspectrum of the TPGS-MWCNTs shows the G band around1565 cm�1 and D band around 1310 cm�1. The shifting of the G and

Table 1Physicochemical characterization of different MWCNTs complexes (Values repre-sented as mean � SD; n ¼ 3).

Formulations Particle size(nm)

Polydispersityindex

% Entrapmentefficiency

DOX/MWCNTs 230.41 � 1.3 0.27 � 0.010 92.5 � 2.62DOX/TPGS-MWCNTs 250.18 � 5.5 0.32 � 0.008 97.2 � 1.20

Fig. 2. Transmission Electron Microscopic images: (A) Pristine, (B) Oxidized, and (C) TPGS conjugated MWCNTs.

Fig. 3. Raman spectra of (A) Pristine, (B) Oxidized, and (C) TPGS/MWCNTs.



D band toward lower Raman shift in TPGS-MWCNTs is mainly dueto increase in the extent of conjugation of TPGS with surfaceengineered MWCNTs, which would increase the single bond char-acteristics in the functionalized system.

X-ray diffraction analysis (XRD) is another valuable tool forcharacterizing the MWCNTs and surface functionalization, whichdepicts the geometry or shape using X-rays. It is a non-destructiveanalytical technique based on the elastic scattering of X-rays. It re-veals information about the crystallographic structure, chemicalcomposition, physical properties and degree of crystallinity [22,40].The Fig. 4 shows the XRD pattern of the pristine, oxidized and TPGSconjugated surface engineered MWCNTs. The XRD analysis of func-tionalizedMWCNTs and TPGS-MWCNTs clearly shows that therewasno change in the seamless tubular structure and were found similarwith procured pristine and purified MWCNTs. Jain & co-workerspreviously reported the XRD pattern of the pristine and surfacefunctionalized MWCNTs and our results are in agreement [17,22].

The drug loading efficiency and in vitro release behavior are themost important, prerequisite characteristic in the development andcharacterization of targeted drug delivery system. The drugentrapment efficiency was studied following a modified dialysisdiffusion technique and was found to be significantly higher forTPGS-MWCNTs than pristine MWCNTs at different ratio of theMWCNTs: drugs. The starting ratio of MWCNTs: DOX was 1:0.5 (w/w), which increased upto 1:4 (w/w) on varying DOX concentration.The anthracycline antibiotic DOX was loaded by incubating withpristine and TPGS-MWCNTs nanoconjugates as evidenced by red-dish color forming “forest scrub” like complex [41]. DOX entrapmentwas monitored by UV/Visible absorption to confirm indirectly, thedrug entrapment in the pristine and TPGS-MWCNTs, during whichthe characteristic absorption peak of DOX at 480.2 nm was slightlyshifted, indicative of interaction between DOX and MWCNTs.

A significant improvement in the entrapment efficiency wasobserved from 92.5 � 2.62 (pristine MWCNTs) to 97.2 � 2.50 (DOX/

Fig. 4. X-ray diffraction (XRD) analysis; (A) Pristine, (B) Oxidized, and (C) TPGS conjugated MWCNTs.


ADMIN

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TPGS-MWCNTs). Due to its aromatic structure the DOX tends tostrongly interact with sidewall and ends of the nanotubes throughpep stacking and hydrophobic interactions. In this, large p-conju-gated structure could form pep stacking interaction with quininepart of the DOX although amino (eNH2) and several hydroxyl (eOH)groups are also present in the chemical structure of DOX. Thus, theeOH and eCOOH functional groups of the nanotubes form stronghydrogen bond with eOH and amino eNH2 groups in DOX. Anotherpossible reason for increased entrapment efficiency may be that thecationically charged DOX molecules could easily get adsorbed withlower potential of TPGS-MWCNTs than pristine MWCNTs via elec-trostatic interaction as well as p-p stacking interaction. Very highDOX loading efficiency mainly depends on the physical properties ofthe nanotubes. Lu & co-workers reported 96% DOX loading efficiency(the weight percentage of initial DOX bound to MWCNTs) in folateconjugated magnetic multi walled carbon nanotubes (FA-MN-MWCNTs) due to strong pep stacking interactions [36]. The in vitrorelease profile of any targeted drug delivery systems is a crucialparameter for determining their overall clinical pharmaceutical ef-ficacy. We investigated the cumulative DOX release from DOX/TPGS-MWCNTs and DOX/MWCNTs nanoformulations at temperature 37 �Cupto 200 h in the phosphate buffer solution (pH 7.4) and sodiumacetate buffer solution (pH 5.3), corresponding to physiological andendosomal pH of the cancerous cells, respectively. The pH of thecytosol is neutral to mildly alkaline (7.4e7.8), while lysosomal pH isacidic (4e5.5) [26,27,34]. The obtained cumulative DOX releasepattern exhibited a non-linear release profile characterized by rela-tively initial faster release followed by sustained and slower releasein later period. The DOX release was found to be 98.5 � 1.45,38.9� 0.85 and 88.3�1.57, and 19.2�1.86 for pristineMWCNTs andTPGS-MWCNTs at pH 5.3 and 7.4, respectively in 24th h. The DOXrelease at pH 5.3 from pristine MWCNTs was not detected after24th h time point. The amount of drug released at 200th h at pH 7.4was 64.3 � 2.39 and at pH 5.3 was 92.02 � 2.57% (Fig. 5). The DOXrelease from surface engineered nanotubes critically depends on pHand solubility of the drug in particular medium and follows the orderof release at all pH range:

DOX=TPGS�MWCNTs > DOX=MWCNTsðSustained Release > Faster ReleaseÞ

The pH-triggered DOX release pattern will be even more bene-ficial if the functionalized-MWCNTs following the intracellular

internalization, trafficking to the lysosomes and lower pH value oflysosomes will augment drug release inside the acidic microenvi-ronment. At this low pH, the hydrophilicity of DOX increases due tothe protonation of the NH2 group native to its structure. Theincreased hydrophilicity aids in overcoming the interaction (pep)among the DOX and the functionalized MWCNTs while facilitatingits detachment from the nanotubes. The release of DOX from thesurface engineered MWCNTs is pH-triggered with slow andcontrolled manner at different pH value [14,26,27,34,36,42].

Stability studies of the optimized pharmaceutical drug productwere performed as per ICH guidelines. The nanoformulations werefound to be the most stable at 5 � 3 �C in dark. No turbidity wasseen in the formulation stored at 5�3 �C and 25 � 2 �C of MWCNTsformulation in dark condition. However, after storage in light at25 � 2 �C, slight turbidity was observed in all the nanotubes for-mulations, which might be due to aggregation of nanotubes whenstored both in light and dark condition at this temperature. At40 � 2 �C, all the formulations showed higher turbidity, which maypossibly be due to formation of larger aggregates (Table 2).

The drug leakage from the developed nanotubes formulationwas performed for a period of sixmonths and found to be negligibleat 5�3 �C. In general, all the developed nanotubes formulationswere found to be most stable at 5�3 �C temperature in dark(Table 2). The lesser drug leakage may be ascribed to the surfaceengineering of the nanotubes, which provide stronger interactionto the nanotubes. These developed nanotubes formulations arecapable to resist the accelerated stress condition and retain guestmoieties of the molecules even at the higher or elevated temper-ature upto six months.

The hemolytic toxicity of pristine MWCNTs was enough to limitits use as drug delivery system. The hemolytic toxicity of DOX,pristine MWCNTs, DOX/MWCNTs, and DOX/TPGS-MWCNTs nano-formulations was studied and compared to assess the effects onerythrocytes (RBCs) in the blood on administration. The percenthemolysis data of free DOX (15.7 � 0.42), pristine MWCNTs(18.0 � 0.50), oxidized MWCNTs (15.5 � 0.55), DOX/MWCNTs(14.6 � 0.28) and DOX/TPGS-MWCNTs (7.50 � 0.18) werecompared. Pristine MWCNTs showed (18.0 � 0.5) highest hemo-lytic toxicity due to the presence of some metallic impurities.However, DOX loaded TPGS-MWCNTs drastically reduced hemo-lytic toxicity. It is clear from the hemolysis toxicity studies that thedegree of functionalization reduces the erythrocytes toxicity. Theseresults are in good agreement with the earlier report [5,14,17].

Fig. 5. Cumulative amount of DOX released from the DOX/MWCNTs and DOX/TPGS-MWCNTs nanoconjugates at 37 � 0.5 �C in phosphate buffer solution (pH ¼ 5.3 and 7.4). (n ¼ 3).



The Methylthiazole tetrazolium (MTT) cytotoxicity assay wasperformed to determine the cancer targeting propensity of the DOXladen developed optimized nanotubes formulation using MCF-7(human breast cancer; derived from pleural perfusion) cell lineand compared with pristine MWCNTs and free DOX solution. Thecytotoxicity assay clearly demonstrated that upon increasing theconcentration from 0.001 to 100 mM the percent viability of thecancerous cells was decreased owing to apoptosis by intercalatingDOX (anthracycline antibiotic) with DNA (Fig. 6; SupportingAnimation). Our cytotoxicity results are in accordance with theprevious published reports [14,24,41]. Similarly, Gu & co-workersreported the IC50 value for SWCNTs-HBA-DOX and SWCNTs-DOXin HePG2 cells to be 4.8 and 7.4 mM, respectively [43].

In next cell culture studies, we determined the DNA content andmechanism of cell death by cell-cycle phase distribution using flowcytometry of the nanotubes formulation. The cell cycle analysiscould be recognized by the four distinct phases in a proliferatingcell population: G1-, S- (DNA synthesis phase), G2- and M-phase(mitosis), while G2- and M-phase have an identical DNA contentand could not be discriminated on the basis of the DNA content[44]. The apoptotic dead cells as well as fragmented nuclei wereshowed in sub-G1 cells. Additionally, sub-G1 population alsoindicated the apoptotic-associated chromatin degradation [29,45].The control cells which were not treated with the DOX, showed27.99%, 29.17% and 42.84% population in the G1, G2 and S-phase,respectively. According to Fig. 7, the percentage of cells in the G1phase increased to G2 27.99%, 24.35%, 76.97% and 86.50% for con-trol, DOX, DOX/MWCNTs and DOX/TPGS-MWCNTs, respectivelyafter 24 h exposure. The majority of the cancerous cells were

arrested in the G1 phase (86.50%), whereas only 5.35% in S-phase incase of DOX/TPGS-MWCNTs nanoformulation. It is notably indi-cated that the doxorubicin, especially DOX/TPGS-MWCNTs attackand induce cell cycle arrest in the G1 phase by intercalating theDNA synthesis and get accumulated in the nucleus with in shortspan in high quantity.

The qualitative and quantitative cellular uptake of DOX wasstudied (Fig. 8 FA, FB, FC, and FD, A, B, C and D). Fig. 8 FD showshigher red fluorescence intensity as compared to other formulationand control group. The doxorubicin has red auto-fluorescence asobserved in Fig. 8. Similarly, higher fluorescence intensity wasobserved for DOX/TPGS-MWCNTs (78.72%), as compared to DOX/MWCNTs (62.46%) and free DOX (58.15%) in R2 region. while con-trol without DOX showed 69.16% fluorescence intensity in R1 re-gion. The observed higher fluorescence intensity clearly suggeststhe higher uptake of the DOX/TPGS-MWCNTs formulations,possibly due to the receptor-mediated endocytosis as well nano-needle specific mechanism (Fig. 8).

The overall pharmaceutical targeting efficacy of the DOX loadedsurface engineered CNTs nanoformulations were evaluated in Balb/c mice. The plasma concentration profiles of the DOX after i.v.administration of the free DOX, DOX/MWCNTs and DOX/TPGS-MWCNTs (5.0 mg/kg body weight) and pharmacokinetic parame-ters were determined using non-compartment modeling as sum-marized in Table 3 (Fig. 9). The area under the curve (AUC0�N) andarea under the first moment curve (AUMC0�N) were calculated tobe 9.3292, 22.3127, 46.4690 and 22.0079, 149.8770 and 937.5830for free DOX, DOX/MWCNTs and DOX/TPGS-MWCNTs, respectively.The AUC(0�N) and AUMC(0�N) of DOX/TPGS-MWCNTs wereapproximately 5.0 and 6.25 folds higher, respectively as comparedto free DOX. The elimination half-life (t1/2) of DOX/TPGS-MWCNTs,DOX/MWCNTs and free DOX was found to be 12.1651, 4.7022 and1.5648, while MRT was found to be 18.1848, 6.5381 and 2.3590,respectively.

The average half-life (t1/2) of DOX/TPGS-MWCNTs (12.1651) was2.58 and 7.8 times, while MRT was 2.78 and 7.7 times longer ascompared to DOX/MWCNTs and free DOX, respectively. The ob-tained results are ascribed to biocompatibility of surface engi-neered nanotubes upon TPGS conjugation allowing longerresidence time inside the body because TPGS render more hydro-philicity and stealth character to nanotubes.

The PEGylated SWCNTs significantly increased the blood circu-lation of the anticancer drug [46]. The obtained results also supportthe extended residence time and sustained release profile of theDOX loaded surface engineered nanotubes formulations in body ascompared to DOX solution and the maximum being for TPGS con-jugated nanotubes formulations. It clearly evinces the improved

Table 2Accelerated stability studies for the DOX/TPGS-MWCNTs formulations.

Stability parameter DOX/TPGS-MWCNTs

Dark Light

T1 T2 T3 T1 T2 T3

Turbidity � � þþ þ þþ þþþPrecipitation � � þþ þ þþ þþChange in color � � þ þ þ þþCrystallization � � þ þ þ þþChange in consistency � � þ þ þ þþPercent drug leakage1 month 0.4 � 0.02 1.2 � 0.04 2.4 � 0.4 1.2 � 0.04 1.6 � 0.06 5.4 � 0.062 month 1.2 � 0.03 1.4 � 0.09 2.6 � 0.03 1.6 � 0.06 1.8 � 0.09 5.8 � 0.244 month 1.6 � 0.12 1.8 � 0.24 3.4 � 0.32 1.8 � 0.22 2.6 � 0.34 6.2 � 0.826 month 1.8 � 0.16 2.2 � 0.18 3.8 � 0.27 2.0 � 0.33 2.8 � 0.42 6.8 � 0.14

T1, T2 and T3 represent 5 � 3, 25 � 2, and 40 � 2 �C temperatures, respectively.All the values represented as mean � SD (n ¼ 3). “e, þ, þþ and þþþ” indicate no change, small change, considerable change and major change, respectively.

Fig. 6. Percent cell viability of MCF-7 cell after treatment with free DOX, DOX/MWCNTs and DOX/TPGS-MWCNTs at 24 h (n ¼ 3).



Fig. 7. DNA content and cell cycle analysis of the DOX and developed MWCNTs formulations on MCF-7 cell lines using flow cytometry. Cells were incubated with the formulation and analyzed by flow cytometry. Cell cycle resultsdisplayed as a histogram. Dip G1: proportion of cells in G0/G1 phase; Dip G2: proportion of cells in G2 phase; Dip S: proportion of cells in S phase. Peaks corresponding to G1/G0, G2/M, and S phases of the cell cycle were indicatedwhere *p � 0.05 vs. control.

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pharmacokinetics with better bioavailability and more prolongedretention in systemic circulation than that obtained on adminis-tration of drugs-MWCNTs and free drug solutions to mice.

A comparative tissue biodistribution study was performed toassess the amount of DOX after i.v. administration of the developednanotubes formulations and highest DOX level was found in tumor,liver and kidney in 24 h in case of DOX/TPGS-MWCNTs. The freeDOX accumulates progressively in liver, where upto 26.76 � 0.14%of dose is localized after 1 h administration whereas only16.43� 0.056% of DOX was found in liver after 24 h. In case of DOX/TPGS-MWCNTs amount of drug accumulated in liver of its initialdose was found to be 38.72 � 0.54 and 28.88 � 0.98 at initial 1stand 24th h (Fig. 10).

The amount of DOX in body depends upon its distribution,metabolism and excretion. Initially the free DOX content in liverwas found to be highest and later it declined in case of free DOXsolution, which might be due to the rapid drug elimination fromliver i.e. prime site of its necessary action. The amount of DOX wasestimated in the tumor organs and was found to be 9.88 � 0.12,12.75 � 0.11, 14.98 � 0.12 and 18.72 � 0.66 at 1, 6, 12, and 24 h,respectively from the DOX/TPGS-MWCNTs formulations. The highlevels of the surface engineeredMWCNTs were found at initial timepoint of administered dose in kidney and the rapid decline in theoverall formulation thereafter indicating that most of the nano-tubes were eliminated through the renal excretion route [14,47]. Itwas inferred that TPGS appended nanotubes formulations were

highly accumulated in tumor-rich organs with sustained drugrelease.

The obtained pharmacokinetic and tissue/organ biodistributiondata of the DOX laden TPGS MWCNTs formulations are in goodagreement with the previous published reports from our[5,9,12,14,16,17,22] and laboratory [26,27,34,47,48]. The in vivo tu-mor targeting efficacy was assayed on MCF-7 tumor bearing Balb/cmodel and the starting tumor size was approximately 100 mm3 forall dose receiving groups of the developed nanoconjugates as wellas normal saline and control group. The tumor volume (mm3) was107.4� 3.5,103.8� 3.9, 99.2� 3.4 and 89.5� 2.84 in case of normalsaline, free DOX, DOX/MWCNTs and DOX/TPGS-MWCNTs at 5thday. The size of the tumor volume (mm3) was reduced to85.9� 2.92 and 45.6� 2.35 in 30th days after treatment with DOX/MWCNTs and DOX/TPGS-MWCNTs formulations, respectively(Fig. 11).

The percentage body weight changes of theMCF-7 bearing Balb/c mice after intravenous injection of the DOX loaded formulationswas calculated upto 30 days. It clearly suggests that the developedformulations does not affect the body weight of the Balb/c mice,while in case of the normal saline treated group the loss of bodyweight was observed.

KaplaneMeier survival curves based on survival time wereplotted for different groups of animals using Log-rank test. Thecurves suggested that the median survival time for tumor bearingmice treated with DOX/TPGS-MWCNTs (44 days; p < 0.001) was

Fig. 8. Qualitative and Quantitative cellular uptake of the DOX in MCF-7 cell: (FA & A) Control, (FB & B) Free DOX solution, (FC & C) DOX/MWCNTs, and (FD & D) DOX/TPGS-MWCNTsformulations (where p � 0.001 vs control).

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

Parameters Cmax (mg/mL) HVD (h) AUC(0�t) (mg/mL h) AUC(0�N) (mg/mL h) AUMC(0�t) (mg/mL h2) AUMC(0�N) (mg/mL h2) t1/2 (h) MRT (h)

Free DOX 6.11 0.36564 9.3066 9.3292 21.6860 22.0079 1.5648 2.3590DOX/MWCNTs 6.06 0.8329 22.3127 22.9233 131.0810 149.8770 4.7022 6.5381DOX/TPGS-MWCNTs 6.52 1.4356 46.4690 51.5587 603.9520 937.5830 12.1651 18.1848

Probability p � 0.05; standard deviation < 5%.Abbreviations: Cmax, peak plasma concentration; t1/2, elimination half life; MRT, mean residence time; AUC(0�N), area under plasma drug concentration over time curve; HVD,half value duration.



extended compared to DOX/MWCNTs (23 days), free DOX (18 days)and control group (12 days) (Fig. 11).

These results further confirmed that the higher tumor treatmentpotential possessed by the surface engineered MWCNTs; resultedin longer survival span for tumor bearing mice. The longest survivalspan (44 days; p < 0.001) was observed in case of DOX/TPGS-MWCNTs. The obtained results were similar to tumor inhibitiongrowth studies. The median survival span for doxorubicin loadednanotubes formulations was found in the following order:

DOX=TPGS�MWCNTs > DOX�MWCNTs > free DOX

The DOX/TPGS-MWCNTs (targeted stealth; long circulatory na-ture) were found to be more active than pristine MWCNTs and drugsolutionwith a significant reduction in tumor growth. Although thefree DOXwas initially efficient in suppressing further tumor growthbut the inhibition activity did not last long. The median survival ofDOX loaded angiopep conjugated MWCNTs (DOX-O-MWCNTs-ANG) was observed 43 days while DOX-O-MWCNTs-PEG showedonly 36 days [30].

Hematological parameters (RBCs, WBCs and differential counts)were determined to investigate the relative effects of the MWCNTsformulations (DOX/MWCNTs, DOX/TPGS-MWCNTs) and comparedwith free DOX on the different components of blood. Blood sampleswere analyzed for RBCs, WBCs and differential counts are shown inTable 4.

The RBCs and WBCs counts of free DOX, MWCNTs, DOX/MWCNTs, and DOX/TPGS-MWCNTs formulations were found to be7.8 � 0.34 � 106/mL, 7.5 � 0.56 � 106/mL, 7.7 � 0.88 � 106/mL,9.1 � 0.22 � 106/mL and 9.5 � 0.46 � 106/mL, 8.6 � 0.62 � 106/mL,8.8 � 0.87 � 106/mL and 10.6 � 0.50 � 106/mL, respectively. Thedifferential counts were found to be very similar to control group.The hemoglobin (Hb) and HCT counts of the DOX/TPGS-MWCNTswere found to be 12.2 � 0.22 (g/dl) and 34.8 � 0.54 and werealmost similar with the control group 12.4 � 0.33 (g/dl) and35.5 � 0.65, respectively.

These obtained data from the serum biochemical parametersclearly suggest that the RBCs, WBCs and differential count of DOX/TPGS-MWCNTs were almost similar with the control and normalsaline groups. Similarly, the differential counts i.e. leucocytes,

Fig. 10. Biodistribution of DOX after intravenous administration of DOX solution, DOX/MWCNTs and DOX/TPGS-MWCNTs formulation in tumor bearing Balb/c mice at a single dose(5.0 mg/kg body weight). *p � 0.05; **p � 0.01; ns: not significant Vs. Free DOX. (Values represented as means � SD; n ¼ 3).

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0 10 20 30 40 50

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Fig. 9. Serum concentration of DOX from free DOX, DOX/MWCNTs and DOX/TPGS-MWCNTs at different data points. Mean � SD (n ¼ 3; p � 0.001).



monocytes and lymphocytes of DOX/TPGS-MWCNTs were almostsimilar to normal values. The hematological values clearly indicatethe superior non-toxic behavior of the TPGS appended than thepristine MWCNTs and free DOX.

The DOX solution induced toxicity was observed in mice as re-flected by the increased activity of SGPT (35.2 � 2.6 IU/L), ALP(69.8 � 2.3 IU/L) and total bilirubin concentration (0.62 � 0.18 mg/100 mL). Upon entrapment of DOX in the pristine MWCNTs a smallincrease in the activity of SGPT (25.6 � 1.9 IU/L), ALP (61.2 � 2.4 IU/L) and total bilirubin concentration (0.46 � 0.020 mg/100 mL) wasobserved. The surface engineering of MWCNTs with subsequentialstep and conjugation of the TPGS have much reduced the in vivotoxicities of the drug loaded formulations.

4. Conclusion

The surface engineered carbon nanotubes have been continu-ously attracting escalating attention in targeted drug delivery. Inthe present studies, TPGS appended stealth surface engineeredMWCNTs nanoformulation was found to be significant in tumorgrowth suppression as compared to non-targeted CNTs and freeDOX solution. The quantitative biodistribution studies furthercorroborated the anti-tumor activities, survival span whereinhigher amount of DOX was found from MWCNTs at the site ofcancerous tissue. The pharmacokinetics studies clearly revealedthat the developed MWCNTs formulations are long-circulating(stealth). Significant reduction in subsequent growth in tumor

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Normal saline Free DOX DOX/MWCNTs DOX/TPGS-MWCNTs

Fig. 11. KaplaneMeier survival curves of MCF-7 bearing Balb/c mice and analyzed by Log-rank (Mental-Cox) test with normal saline group as control. *p � 0.05; **p � 0.01; ns: nosignificant difference (Upper). Tumor regression analysis after intravenous administration of free DOX, DOX/MWCNTs and DOX/TPGS-MWCNTs nanoconjugates (5.0 mg/kg bodyweight dose). The DOX/TPGS-MWCNTs treated group showed significant (P � 0.05) suppression of tumor growth compared with the other groups (lower) (n ¼ 3).

Table 4Serum biochemical parameters of Balb/C mice treated with free DOX, MWCNTs, DOX/MWCNTs and DOX/TPGS-MWCNTs formulation after 7 days.

Group RBCs (�106/ml) WBCs (�106/mL) Differential counts (�103/mL) Hb (g/dl) HCT

Monocytes Lmphocytes Neutrophils

Control 9.2 � 0.40 10.8 � 0.40 1.4 � 0.60 7.9 � 0.42 1.6 � 0.42 12.4 � 0.33 35.5 � 0.65Normal saline 8.4 � 0.32 9.6 � 0.42 0.9 � 0.34 6.1 � 0.44 1.0 � 0.66 10.5 � 0.22 34.4 � 0.25Free DOX 7.8 � 0.34 9.5 � 0.46 0.9 � 0.33 6.1 � 0.42 1.1 � 0.82 10.6 � 0.32 33.8 � 0.62MWCNTs 7.5 � 0.56 8.6 � 0.62 0.7 � 0.76 5.9 � 0.88 0.9 � 0.85 9.8 � 0.94 33.6 � 0.12DOX/MWCNTs 7.7 � 0.88 8.8 � 0.87 0.9 � 0.90 6.1 � 0.62 1.1 � 0.48 10.2 � 0.66 34.0 � 0.23DOX/TPGS-MWCNTs 9.1 � 0.22 10.6 � 0.50 1.2 � 0.65 7.8 � 0.92 1.5 � 0.4 12.2 � 0.22 34.8 � 0.54

All values are expressed as mean � SD. No. of animals per time points were three (n ¼ 3); WBCs, white blood corpuscles; RBCs, red blood corpuscles; Hb, haemolglobin; HCT,haematocrit.



was probably due to ligand-driven internalization of the surfaceengineered nanotubes targeted drug delivery system at the targetsite/tissues, which was accompanied by slow and sustained releaseof the drug. The tumor growth inhibition study clearly indicatedthat inclusion of the pH-responsive characteristics increases theoverall pharmaceutical targeting efficiency of the targeted nano-tubes formulations. To the best of our knowledge, the present one isa debut study report and suggest that DOX/TPGS-MWCNTs is apromising and efficient targeted drug delivery system in thetreatment of cancer.

Declaration of interest

The authors report no conflict of interest.

Acknowledgments

One of author Neelesh Kumar Mehra is highly thankful to Uni-versity Grants Commission (UGC), New Delhi for providing theSenior Research Fellowship during the tenure of the studies. Theauthor also acknowledge Dr. Ranveer Kumar Department of Phys-ics, Dr. H. S. Gour University, Sagar for Raman spectroscopy; CentralInstruments Facilities (CIF), National Institute of PharmaceuticalEducation and Research (NIPER), Mohali, Chandigarh; Central DrugResearch Institute (CDRI), Lucknow; All India Institute of Medicineand Sciences (AIIMS), New Delhi (TEM); Diya Laboratory, Mumbai.Authors are also thankful to National Centre for Cell Sciences(NCCS), Pune for providing the cell lines.

Appendix A. Supplementary data

Supplementary data related to this article can be found online athttp://dx.doi.org/10.1016/j.biomaterials.2014.02.022.

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