Augmented Anticancer Activity of a Targeted, Intracellularly Activatable, Theranostic Nanomedicine Based on Fluorescent and Radiolabeled, Methotrexate-Folic Acid-Multiwalled Carbon

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Article

Augmented Anticancer Activity of a Targeted, Intracellularly Activatable,Theranostic Nanomedicine based on Fluorescent and Radiolabeled,

Methotrexate-Folic acid-Multiwalled Carbon Nanotube ConjugateManasmita Das, Satyajit R. Datir, Raman Preet Singh, and Sanyog Jain

Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp300701e • Publication Date (Web): 17 May 2013

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Augmented Anticancer Activity of a Targeted, Intracellularly

Activatable, Theranostic Nanomedicine based on Fluorescent and

Radiolabeled, Methotrexate-Folic acid-Multiwalled Carbon

Nanotube Conjugate

Manasmita Das, Satyajit R. Datir, Raman Preet Singh, Sanyog Jain*

Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of

Pharmaceutical Education and Research (NIPER), Sector 67, SAS Nagar (Mohali) Punjab INDIA160062

*Corresponding author.

Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics

National Institute of Pharmaceutical Education and Research (NIPER)

Sector 67, SAS Nagar (Mohali) Punjab- 160062 India

Tel.: +91172-2292055, Fax: +91172-2214692

E-mail addresses: [email protected], [email protected] (S. Jain)

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Abstract

The present study reports the design, synthesis and biological evaluation of a novel, intravenously

injectable, theranostic prodrug based on multiwalled carbon nanotubes (MWCNTs) concomitantly

decorated with a fluorochrome (Alexa-fluor, AF488/647), radionucleide (Technitium-99m), tumor

targeting module (folic acid, FA) and anticancer agent (methotrexate, MTX). Specifically, MTX was

conjugated to MWCNTs via a serum-stable yet intracellularly hydrolysable ester linkage to ensure

minimum drug loss in circulation. Cell uptake studies corroborated the selective internalization of AF-

FA-MTX-MWCNTs (1) by folate receptor (FR) positive human lung (A549) and breast (MCF 7) cancer

cells through FR mediated endocytosis. Lysosomal trafficking of 1 enabled the conjugate to exert higher

anticancer activity as compared to its non-targeted counterpart that was mainly restricted to cytoplasm.

Tumor-specific accumulation of 1 in Ehlrich Ascites Tumor (EAT) xenografted mice was almost 19 and

8.6 times higher than free MTX and FA-deprived MWCNTs. Subsequently, the conjugate 1 was shown to

arrest tumor growth more effectively in chemically breast tumor induced rats, when compared to either

free MTX or nontargeted controls. Interestingly, the anticancer activities of the ester-linked CNT-MTX

conjugates (including the one deprived of FA) were significantly higher than their amide-linked

counterpart suggesting that cleavability of linkers between drug and multifunctional nanotubes critically

influence their therapeutic performance. The results were also supported by in silico docking and ligand

similarity analysis. Toxicity studies in mice confirmed that all CNT-MTX conjugates were devoid of any

perceivable hepatotoxicity, cardiotoxicity and nephrotoxicity. Overall, the delivery property of

MWCNTs, high tumor binding avidity of FA, optical detectability of AF fluorochromes and radio-

traceability of 99mTc could be successfully integrated and partitioned on a single CNT-platform to

augment the therapeutic efficacy of MTX against FR over-expressing cancer cells while allowing a real-

time monitoring of treatment response through multimodal imaging.

Keywords: Multiwalled carbon nanotubes, folate-receptor mediated endocytosis, cancer, methotrexate,

scintigraphy, tumor-targeted delivery

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1. Introduction

Functionalized carbon nanotubes (CNTs) have emerged as one of the most versatile and innovative

nanovectors for drug delivery1, 2. CNTs possess unique physicochemical and structural properties such as

high aspect ratio and surface area, tunable surface chemistry, ultrahigh drug loading capacity via π-π

stacking interactions and photoacoustic effects, which make these nanocarriers an attractive probe for

multifarious biomedical applications including targeted drug delivery and multimodal imaging3-5. With

recent surge of interest in theranostic nanosystems, there have been phenomenal impetuses in the

development of multifunctional CNT-based platforms, concomitantly tethered with multiple chemical

species including biofunctional spacers (PEG), tumor homing agents, therapeutic drugs/genes,

fluorochromes and radionucleides. Such “chemical partitioning” enables CNTs to simultaneously track,

target and treat diseased cells6-11 while allowing a real time monitoring of treatment response through

noninvasive, multimodal imaging.

Over the last decade various covalent approaches of CNT functionalization have been exploited to

develop multifunctional CNT-based platform for theranostic applications. For example, Pastorin et al.

have executed the double functionalization of multiwalled carbon nanotubes (MWCNTs) with a

fluorescent molecule (viz. fluorescein isothiocyanate) and an anticancer agent (viz. methotrexate) and

showed that these bifunctional CNTs are efficiently taken up by Jurkat cells6. Similarly, Heister et al. tri-

functionalized oxidized single walled carbon nanotubes (SWCNTs) with the anticancer drug doxorubicin

(DOX), a monoclonal antibody and a fluorescent marker and demonstrated that these functionalized

nanotubes were efficiently taken up by cancer cells with subsequent translocation of the drug into the

nucleus12. In another notable study, Dhar et al. used folate as a tumor homing device for SWCNT-

mediated Pt (IV) prodrug delivery7. Some recent reports have also demonstrated the feasibility of using

folate conjugated, magnetic multiwalled carbon nanotubes as a dual targeted delivery system for

Doxorubicin13, 14. Despite some recent advancement in the design and fabrication of multifunctional

CNTs, severe limitations still persevere as much of the interaction of functionalized CNTs with living

cells and tissues is still an unraveled mystery. Further, most of the reports on multifunctional CNTs are

restricted to preliminary in vitro investigations. A few studies have embarked on the feasibility of using

antibody/ peptide conjugated SWCNTs/MWCNTs for targeted anticancer drug delivery in vivo15-18.

However, such reports are very limited and in most cases, have dealt with relatively simpler surfaces

comprised of double- and even monofunctional CNTs. Moreover, the fundamental question of how the

integration of multiple functional entities on the same nanotube platform influences their targeting and

therapeutic efficacy in vivo has not been adequately addressed in many of these reports. The present

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study, in principle, was motivated by the interest to improve our fundamental understandings on the effect

of “functionalization partitioning” on the in vivo behavior of CNTs. In line with that approach, we

designed a novel, intravenously injectable, receptor-targeted prodrug based on folate-methotrexate (FA-

MTX) co-conjugated MWCNTs. While the FA moiety on CNTs facilitate easy detection of cancer cells

via ligand-receptor binding affinity interactions, MTX can regress the cancer cells by inhibition of

dihydrofolate reductase (DHFR), a key enzyme responsible for FA biosynthesis. This therapeutic

conjugate was further labeled with a fluorescent dye, Alexa-Fluor (AF-647/488) and a radio-tracer,

Technitium-99m (99mTc) to facilitate the instantaneous tracking of intracellular trafficking and bio-

distribution of the nanovector through combined optical imaging and radioscintigraphy.

Multifunctionalization of MWCNTs with FA, MTX, AF-fluorochrome and 99mTc led to the formation of a

multimodal, theranostic nanoprobe, capable of performing concomitant detection, regression and imaging

of folate receptor (FR)-over-expressed cancer cells. In this regard, it is worthy to mention that all

chemical species were introduced on the surface of MWCNTs through distinct chemical linkages so that

their stability and activity in biological systems can be coordinated in an optimal fashion. Specifically,

MTX was conjugated to MWCNTs via an intracellularly hydrolysable yet serum stable ester linkage

whereas FA and AF-647 were coupled through relatively robust, hydrolytically stable amide linkages.

Likewise, 99mTc was coordinated with MWCNTs through either free -NH2 donor or negatively charged

hydroxyl (–O-)/ carboxyl (-COO-) groups on their surface. Taking this tetrafunctional CNT platform as a

model, we tried to elucidate whether and how the various functional molecules associated with CNTs

influence their cell internalization, biodistribution and anticancer efficacy. To the best of our knowledge,

this is the first example wherein acid-oxidized, carboxylated MWCNTs have been tetrafunctionalized

with a fluorochrome, targeting ligand, chemotherapeutic agent and radio-tracer to facilitate multimodal

imaging and molecularly targeted therapy in vivo while avoiding deleterious side-effects to normal cells.

2. Materials and methods.

2.1. Materials

Pristine (p) MWCNTs (purity > 95%, length 1-2 µm and diameter 20-30 nm) were procured from

Nanovatech Pvt. Ltd., U S. Sulphuric acid, nitric acid (69-72%), disodium hydrogen phosphate, sodium

acetate, thionyl chloride, sodium lauryl sulphate, copper sulphate and thiobarbituric acid were purchased

from Loba Chemie Pvt. Ltd., Mumbai, India. Methotrexate was obtained as gift sample from Fresenius

Kabi Oncology Limited, Gurgaon India. 2, 2’-(ethylene dioxy) bis-(ethylene amine), folic acid, glycidol,

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium], 4′,6-diamidino-2-phenylindole

dihydrochloride (DAPI) and 7,12 Dimethylbenz [α]anthracene (≥ 95% pure) were purchased from

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Sigma, USA. 7,12-dimethylbenz [α]anthracene (DMBA; ≥ 95% pure), 2, 2’-(ethylene dioxy) bis-

(ethylene amine) (EDBE), were purchased from Sigma, USA. All kits for biochemical estimations were

procured from Accurex, Biomedical Ltd, Mumbai. Culture medium and serum were procured from PAA,

Austria. A549 cells were obtained from the National Centre for Cell Sciences (NCSS). Maleimide ester of

AF-647/488 was purchased from Invitrogen, USA. All other chemicals and solvents were of analytical

grade and procured from local suppliers unless otherwise mentioned.

2.2. Methods

2.2.1. Tetrafunctionalization of MWCNTs

Tetrafunctionalization of MWCNTs with AF-647, FA, MTX and 99m Tc was executed in a number of

steps, as schematized in figure 1. Acid oxidized carboxylated MWCNTs were prepared by 3h oxidation

of p-MWCNTs in presence of mixed acids using the protocol described in our earlier reports19 . These

carboxylated MWCNTs were conjugated to AF-647, FA, MTX and 99m Tc using the protocol detailed as

follows.

2.2.1.1. Amine functionalization of MWCNTs

For amine functionalization, oxidized-MWCNTs (100 mg) were dispersed in DMF (5 ml) via

ultrasonication for 5 minute. To the resultant dispersion, SOCl2 (15-20 ml) was added and the mixture

was refluxed at 80°C for 24 h20. Thereafter, solvents were removed using rotavapor and the resultant

oxidized MWCNTs were dispersed in anhydrous THF. From thermo-gravimetric analysis (TGA),

carboxylic density on the surface was determined to be 0.0018 mmoles/mg of CNTs. This value was

necessary to determine the stoichiometry of amine functionalization reaction. Based on TG results,

acylated MWCNTs suspended in a 5:1 (v/v) mixture of DMSO and pyridine were flooded with

approximately 5 fold molar excess of EDBE dissolved in anhydrous DMSO21, 22. The reaction mixture

was left to stirring for 12 h, following which the reaction mixture was subjected to centrifugation and

repeated washings with water (3×10 ml) and acetone (2× 10 ml) to free the aminated MWCNTs from

unreacted reagents and biproducts. Surface amine density of functionalized MWCNTs was determined

using the p-nitrobenzaldehyde, colorimetric assay using a previously published protocol23. Yield: 95%

(w/w), black powder.

2.2.1.2. Conjugation of AF and FA with amine functionalized MWCNTs

As a preliminary step towards the conjugation of AF-647 and FA with 2, N-hydroxysuccinimide (NHS)

ester of FA was prepared using standard carbodiimide chemistry following our previously reported

protocol24. Subsequently, FA-NHS ester (0.5 µmol) was dissolved in freshly distilled DMSO (1 ml) and

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added to a suspension of amine-MWCNTs (20 mg) in water (5 ml). To the suspension, maleimide ester of

AF-647 (0.05 µmol), dissolved in a 1:1 (v/v) mixture of THF:H2O was added and the reaction mixture

was stirred for 24 h in dark. Thereafter, the reaction mixture was subjected to centrifugation and the

supernatant was discarded. The pellet of AF-FA-MWCNTs was washed with distilled water, re-

centrifuged to remove the unreacted dye/ FA-NHS and subjected to freeze drying. Yield: 90% (w/w),

black powder.

2.2.1.3. Glycidylation of FA-MWCNTs

The conjugation of glycidol to FA-MWCNTs was performed according to our previously published

protocol25. Briefly, an ethanolic solution of glycidol (10 ml, 1% v/v) was added dropwise to a colloidal

suspension of AF-FA-MWCNTs with ultrasonication. A few drops of triethyl-amine were added and the

reaction mixture was stirred for 24 h in dark. Glycidylated MWCNTs (MWCNT-AF-FA-GLY) were

recovered by centrifugation, followed by repeated washings with water and acetone.

2.2.1.4. MTX conjugation with glycidylated MWCNTs

For conjugation of MWCNT-AF-FA-GLY with MTX, MTX (0.02 mmol) was dissolved in minimum

amount of DMSO and diluted with 10 ml of water. The resulting solution was then mixed with an

aqueous solution of 1-ethyl-3-(3-dimethylamino)-propyl) carbodiimide (EDC) (0.03 mmol) and N-

hydroxy succinimide (NHS) (0.03 mmol). The pH of the solution was then adjusted to ~8 by dropwise

addition of triethyl amine. An aqueous dispersion of ~25 mg of glycidylated nanotubes was added to the

reaction mixture and stirring was continued for additional 24h at 37ºC in the dark. Thereafter, the

trifunctional conjugate (1) was isolated via centrifugation, washed 5 times with de-ionized water and

acetone and finally air-dried. Yield: 92% (w/w), black powder.

2.2.1.5. Radio-labeling of AF-FA-MTX-MWCNTs

An aqueous suspension of free AF-FA-MTX-MWCNTs was radio-labeled with 99mTc by direct labeling

method using stannous chloride (SnCl2) as the reducing agent26-28. Briefly, 0.1 ml of sodium pertechnetate

(99mTcO4-, approximately 2µCi, obtained by solvent extraction method from molybdenum) was mixed

with 50 ml of SnCl2 solution (defined concentration to give 25–200 mg of SnCl2 in 50ml) in 10% acetic

acid solution to reduce technetium. The solution pH was adjusted to 6.5–7.0 using 0.5 (M) sodium

bicarbonate solutions. To this mixture, 1ml of nanotube suspension (1mg/ml in H2O) was added and

incubated for 15 min at room temperature. This procedure often leads to the formation of radio colloids

(reduced and hydrolyzed Tc-99m, TcO2) that were separated from the radio-labeled formulations by

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centrifugation, followed by washing with normal saline. The purified radio-labeled formulations were

stored in sterile evacuated sealed vials for subsequent studies. The same method was used for

radiolabeling of free MTX and CNT-MTX conjugates used as a control for in vitro and in vivo studies.

2.2.1.6. Synthesis of control conjugates

In order to comprehend the influence of surface functional molecules and linkers on the in vivo behavior

of MWCNTs, two control conjugates were synthesized: (i) FA-MTX co-tethered MWCNTs in which

MTX was conjugated to MWCNTs via a relatively stable amide bond (2) and (ii) FA-deprived MWCNT-

MTX conjugates in which MTX was conjugated to CNTs via ester bond (3). For synthesis of amide-

linked conjugate, AF-647/488 ester (0.05 µmol), FA-NHS ester (0.5 µmol) and MTX-NHS ester (0.5

µmol) were separately dissolved in DMSO and added sequentially to an aqueous suspension of amine

functionalized MWCNTs (20 mg). The reaction was stirred for 24h in dark following which the pellets of

AF-FA-MTX-MWCNTs (amide linkage) was collected by centrifugation and freed from unreacted

materials/biproducts through repeated washing with water and acetone. For preparation of FA-deprived,

ester-linked CNT-MTX conjugate, amine functionalized MWCNTs were glycidylated using the same

protocol described in Section 2.2.1.3. An equivalent concentration of MTX used in the reaction of

MWCNT-AF-FA-GLY with MTX was coupled with glycidylated CNTs using the same protocol

described in Section 2.2.1.4. A PEGylated control (4) was synthesized in which carboxylated MWCNTs

were acylated and reacted with m-PEG 5000, following the same protocol used for amine

functionalization of MWCNTs. The chemical structures of all CNT conjugates including controls have

been schematized in Figure 2.

2.2.2. Physicochemical characterization of synthesized conjugate bound to MWCNTs

Size and morphology of f-MWCNTs were analyzed using scanning electron microscopy (SEM, Model

S30400) and transmission electron microscopy (TEM, Model FEI Tecnai G2). Surface charge and

hydrodynamic sizes and zeta potential measurements were done using the Malvern Zeta Sizer (Nano ZS,

Malvern Instrument, US). Surface chemistry of FA-MTX-MWCNTs (without AF/ 99mTc) was prelimnary

studied using Fourier transform infrared (FTIR) while fine-structure resolved analysis of the surface

bound ligands was performed using high resolution magic angle spinning NMR (HRMAS-NMR)

spectroscopy. FTIR spectra were recorded on Perkin Elmer systems using KBr pellets and processed

using Spectrum Software. TGA was carried out on a Perkin Elmer System by heating 5mg of p and f-

CNT at the rate of 10°C/min. Samples for HRMAS-NMR experiments were prepared by suspending 10

mg of each nanotube preparation in a 1:1 mixture of DMSO-d6: D2O (500 µL)29. HRMAS–NMR analysis

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was carried out with a 400 MHz FT-NMR spectrometer (Avance 400), equipped with a 5 mm HRMAS

probe.

2.2.3. Quantification of functional molecules associated with MWCNTs

The concentration of AF-647/488 on nanotube surface was determined by measuring the optical density

of an aqueous suspension of the nanoconjugate at 647/488 nm. Free AF-647 was used as the reference. To

further determine the extent of FA conjugation on the surface of CNTs, nanotubes were digested with

trypsin at 37°C for 12 h under continuous stirring. Following tryptic hydrolysis of FA-conjugated

MWCNTs, the folate density on MWCNTs was determined by spectrophotometrically by recording the

absorbance of hydrolyte at 358 nm (folic acid = 8643.5 M−1 cm−1). For quantifying the extent of MTX

immobilization on MWCNTs, a suspension of AF-FA-MTX-MWCNTs in PBS was stirred for 24 h in

presence of porcine liver esterase. The pH of the resulting suspension was continuously maintained to 8 in

order to facilitate hydrolysis of the ester bond. Following centrifugal separation of the nanotubes, the

average number of MTX immobilized per particle was determined spectrophotometrically by recording

the absorbance of the hydrolysis solution at 302 nm. The labeling efficiency of 99m Tc labeled MWCNTs

was determined by ascending instant thin layer chromatography (ITLC) methods following our previously

published protocol28.

2.2.5. pH dependent release of MTX from MWCNTs

The release behavior of MTX from AF-FA-MTX-MWCNTs was checked at different conditions: (: (i)

PBS (pH 7.4) (ii) (ii) A549 cell extract iii) rat plasma (pH 7.4) and (iv) pH 4.5 in presence of lysozymes

(to mimic the endosomal conditions). Briefly, aqueous dispersions of MWCNTs (10 mg suspended in 2

ml of water) were filled in the dialysis membranes (MW cut off 12000 Da) and then poured inside release

medium in water shaker bath for 24 h. Aliquots were taken at regular time intervals and replaced with

equivalent volume of buffer. Finally, absorbances of the aliquots were recorded at 302 nm.

.

2.2.6.2. In vitro stability of the radiolabeled formulations

For determination of in vitro stability, 100 µl of the tetrafunctional conjugate/ control was mixed with 2.0

ml of PBS (pH 7.4) and serum (pH 7.4) incubated at room temperature and the change in labeling

efficiency was monitored over a period of 24 h by ITLC as described before.

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2.2.7. In vitro cellular uptake and cytotoxicity studies

2.2.7.1. Cell Culture

In vitro cellular uptake and cytotoxicity studies were conducted in folate receptor (FR) positive human

lung (A549) and breast (MCF 7) adenocarcinoma cell lines30-36. Cells (1x104 cells/well) were grown in

Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum 2mM

glutamine, 100 units/ ml penicillin, 100µg/ml streptomycin, 4mmol/L glutamine at 37°C in a 5% CO2 and

95% air humidified atmosphere. Confluent cultures were harvested by trypsinization, cells counted and

suitably diluted to obtain 5x105 cells/ml. Cell suspension (200 µl) was added in 96 well tissue culture

plates and incubated overnight for cell attachment.

2.2.7.2. Cellular uptake study

For evaluation of intracellular uptake, cultured cells were exposed to 100µg/ml of AF-FA-MTX-

MWCNTs for 3h in absence and presence of excess FA (50µg/ml). Cell uptake was visualized using

confocal microscopy [Model Olympus FV 1000]. Intracellular trafficking of AF-488/ AF-647 labeled

MWCNTs were studied by labeling lysosomes and nuclei of A549 cells with neutral red (NR), and DAPI

respectively as described in our earlier reports22, 24, 37.

2.2.7.3. Cytotoxicity study

For cytotoxicity study 4X105 A549 and MCF 7 cells were seeded to 96 well tissue culture plate in a total

volume of 180µl of complete media and kept for 18h following which aqueous dispersions of 0.1-

100µg/ml of free MTX and different CNTs preparation were added to the cells at different concentrations,

incubated for 1h at 37°C in a humidified incubator (HERA cell) maintained at 5% CO2. After incubation

for 24h, MTT (4µg/ml) was added to each well at the strength of 10% v/v and incubated for further 4h at

37°C. Subsequently the media containing MTT was removed and 200µl of DMSO was added to dissolve

the Formosan crystals. The absorbance was measured using an ELISA plate reader at 595nm 38.

2.2.8. Docking study

Docking studies of free MTX and f-MWCNTs 8 with DHFR (PDB ID=1U72) was performed using

Discovery studio ® while ligand similarity analysis was performed with ROCS.

2.2.9. In Vivo Studies

2.2.9.1.1. In vivo stability 99mTc-AF-FA-MTX-MWCNTs

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In vivo stability of free drug, 99mTc-AF-FA-MTX-MWCNTs and all control formulations was assessed in

normal, healthy, female New Zealand rabbits using our previously reported protocol26.

2.2.9.1.2. Quantitative organ distribution study

Bio-distribution of free drug, 99mTc-AF-FA-MTX-MWCNTs and all control formulations was studied in

folate receptor (FR) positive Ehlrich Ascites Tumor (EAT) bearing mice model. The protocol for organ

distribution study was duly approved by institutional animal ethical committee of Indian Nuclear

Medicine and Allied Science (INMAS). The tumor was implanted into the mice by injecting 0.1 ml of cell

suspension of EAT cells (Ehlrich Ascites Tumor) subcutaneously in right hind paw of the mice. The

injected volume contained approximately 1.0-1.5 X 107 cells. Tumor was allowed to grow for 10 days.

The mice were divided into 4 groups of 12 animals each (total 48 mice, n=3 mice per time point). Each

mouse received 100 µCi (100 µl) doses of the labeled formulations by separate intravenous injection

through the tail vein. Mice were humanely sacrificed at 1, 4 and 24 h after the injection. The blood was

collected by cardiac puncture. Subsequently, different organs like heart, lung, liver, kidney, spleen, bone,

stomach, intestine, tumor and muscle were dissected, washed with Ringer’s solution to remove any

adherent debris and dried using tissue paper. The organs were taken in pre-weighed tubes, which were

weighed again to calculate the weight of organ/tissue and radioactivity corresponding to them was

measured using well-type γ-scintillation counter. The results were expressed as percentage of injected

dose (ID) per gram of an organ.

2.2.9.2. Tumor growth inhibition studies

Antitumor efficacy of free drug, 99mTc-AF-FA-MTX-MWCNTs and all control formulations was studied

in female Sprague Dawley (SD) rats. Tumors were chemically induced in animals using 7, 12-

dimethylbenz[α] anthracene (DMBA) as the carcinogen. Protocols for animal experiments were duly

approved by the Institutional Animal Ethics Committee (IAEC) of NIPER. Rats were divided into 6

groups, each group containing 4 animals. The first 5 groups of animals were intravenously administered

with aqueous dispersions of 5 mg/ kg of free MTX and various f-MWCNTs twice at weekly intervals via

intravenous injection. The last group was kept as control, which received normal saline in a similar way.

The tumor volume and body weight was measured on every alternate day post treatment by vernier

caliper using the following equation: Tumor volume (V) = D*d2/2, where,‘d’ is the smallest and ‘D’ is the

longest length of the tumor. The study was terminated after 15 days post-treatments.

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2.2.9.3. Toxicity Studies

In order to address the toxicity issues pertaining to free MTX as well as CNTs bound to different

chemical species, toxicity studies were carried out in Swiss mice, intravenously injected with free MTX

as well as functionalized MWCNTs at the dose similar to that used in efficacy studies. Toxicity studies

were conducted in mice because compared to rats, mice present more sensitive models for toxicity

evaluation. Female Swiss mice (weighing ~25 g) were procured from Central Animal Facility, NIPER

Mohali. Animals were divided into 6 groups, each group receiving a single dose of free MTX, MWCNT-

AF-647-FA-GLY-MTX, MWCNT-AF-647-FA-GLY, MWCNT-AF-647-FA and MWCNT-EDBE-AF-

647 were administered into the first five groups of mice (n=6) through intravenous injection via tail vein.

The sixth group was kept as control and received normal saline in similar manner. Following intravenous

injection, general health conditions of mice including appetite, activity and body weight were monitored

and recorded at regular interval. At the end of 15 days, blood was collected through cardiac puncture in

heparinised capillary tubes. Plasma, separated by centrifugation at 10000 RCF for 10 min, was stored at -

20ºC until analysis. Thereafter, animals were humanely sacrificed and the individual organs (viz. liver,

spleen, kidney and lungs) were excised and weighed to determine the organ indices. Organ index, which

is a marker of general organ level toxicity, was measured by calculating the ratio of increase in organ

weight due to inflammation and cellular infiltration to the reduction in total body weight. Thereafter,

enzyme activities such as Creatine Phosphokinase (CK-MB), Lactate dehydrogenase (LDH), Aspartate

aminotransfarase (AST), Alanine Transaminase (ALT) and Blood urea nitrogen (BUN) levels were

analyzed in plasma while malondialdehyde (MDA) and superoxide dismutase (SOD) were determined in

heart homogenate by the commercially available kits based on the method provided by the manufacturer

instructions supplied with the commercial kits. The detailed procedure for the determination of various

biochemical markers of hepatoxicity and nephrotoxicity have been detailed in our earlier publication39.

3. Results and Discussions

3.1. Tetrafunctionalization of acid-oxidized MWCNTs

The decoration of MWCNTs with multiple bioactives was accomplished in a number of steps, as depicted

in Figure 1. While both SWCNTs and MWCNTs have shown promise in the field of drug delivery, we

proceeded with MWCNTs because the latter presents a wider surface that permits a more efficient

internal encapsulation and external functionalization with active molecules as compared to their single-

walled counterparts40. Further, in a number of studies focusing on nanotoxicology of CNTs, it has been

found that MWCNTs are less toxic as compared to SWCNTs, which make them an ideal choice for in

vivo applications6, 41. In this study, carboxyl MWCNTs, prepared by mixed acid oxidation19, were used as

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the bio-conjugating precursor. To make the initial part of the synthesis more amenable to large-scale

production, we planned to grow up single structured, homofunctionalized MWCNTs in bulk and then

tune up its surface functionality in a modular fashion (Figure 1). In line with that approach, the carboxyl

functions on the surface of acid-oxidized MWCNTs were interchanged with amine groups through a

sequence of thionyl chloride (SOCl2) acylation and subsequent reaction with excess of EDBE to avoid

cross-linking. In a number of studies, it has been reported that insertion of a small PEG-like spacer

between targeting ligands and nanocarriers not only improve the hydrophilicity of the overall conjugate

system but increases its accessibility towards receptor site as well22, 24, 42. As determined by p-

nitrobnzaldehyde assay, the number of free amine groups on the surface of EDBE-MWCNTs was

sufficient enough (0.458 µmoles/ mg) to ensure high-density multiple ligand grafting on the surface of

nanotubes. We sought to derivatize only 50% of the surface amine groups with AF and FA so that the

remaining half may be available for conjugation with the anticancer drug and radionucleide. Both AF and

FA were conjugated to EDBE-MWCNTs via amide linkages using standard maleimide/ succinimide

linker chemistry. As determined spectrophotometrically, the conjugation efficiency of AF-647 and FA

were determined to be approximately 0.038 and 0.186 µmol per unit mass (mg) of MWCNTs

respectively. The choice of a proper linkage between a toxin and a carrier molecule is crucial to

successful drug delivery and release. Herein, we tried to present an improved design of a theranostic

prodrug in which (i) multiple copies of the therapeutic agent can be accommodated on the same nanotube

platform along with other functional bioactives to exert maximum therapeutic effect and (ii) the drug

molecule will be conjugated to the carrier via a cleavable linker that can be degraded in tumor-specific

low pH environments by dual hydrolytic and enzymatic cleavage. In a number of studies focusing on the

synthesis and biological evaluation of amide or ester-based prodrugs of various anticancer/ anti-

inflammatory compounds, it has been reported that ester linkages are more easily hydrolysable under

physiological conditions and subsequently imparts higher activity compared to their amide based

counterparts43, 44. However, the possibility of drug degradation in plasma through serum esterase activity

cannot be totally ruled out. We, therefore, considered of conjugating MTX to MWCNTs through a serum-

stable yet lysosomally degradable ester linkage. As quoted by some earlier reports, the stability of an

ester, in human serum, can be considerably increased by: (i) introducing substitutent(s) on the α -carbon

of the alcohol or acid; (ii) introducing substituent at C-2/ C-3 of the alcohol; (iii) increasing the length of

the alcohol chain from 2 to 3 carbon atoms; (c) (iv) by increasing the size of the substituent group on the

terminal nitrogen45. We reasoned that chemical transformation of the residual surface amine groups to C-

2/ C-3 substituted, branched alcohols might pave the way to formation of a serum-stable ester prodrug of

CNTs. Subsequently, AF-FA-MWCNTs were reacted with glycidol. Glycidylation converted the residual

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primary amino groups of MWCNT-AF-647-FA to alcohol groups, leading to four hydroxyl groups per

amine function. These hydroxyl groups not only ensured high density covalent attachment of drug

molecules on MWCNTs but also played a crucial role in augmenting the aqueous-dispersibility of the

final formulation. Following AF and FA conjugation, the concentration of residual amine groups on the

surface of MWCNTs was 0.234 µmol/mg. As each amine group was supposed to generate four hydroxyl

functionalities, the maximum theoretically possible hydroxyl density per unit mass of glycidylated

MWCNTs was 0.936µmol/mg. Based on this calculated value, the stoichiometry of MTX conjugation

step was restricted to 0.8 µmol MTX per unit mass (mg) of MWCNTs. Reaction of AF-FA-MWCNTs

(OH) with MTX using standard EDC chemistry afforded the desired trifunctional conjugate (MWCNT-3)

in conspicuous yield (~92%w/w). The efficiency of MTX conjugation was determined to be

approximately 86.39%, which corresponded to a practical loading of 33.8 % (w/w of MWCNTs).

Stoichiometric calculations revealed that MTX density on the surface of AF-FA-MTX-MWCNTs (0.751

µmol/ mg) was nearly thrice the residual amine density (0.234 µmoles/ mg) available after conjugation of

FA and AF-fluorochrome with MWCNTs. These results indicate that glycidylation can be used as a

viable technique for generation of multiplicity on the reactive ends of surface-pendant groups associated

with a solid support. The final synthesis step included radiolabeling of 1 with 99mTc. Although it was not

possible to exactly determine the chemical nature and quantity of the functional group (s) that were

involved in coordination with 99mTc, the residual hydroxyl and/ or amine groups on 1 is believed to form

efficient chelates with 99mTc. As described in our earlier reports, 99mTc was coordinated to MWCNTs by

direct labeling method using SnCl2 as the reducing agent26-28. 99mTc was chosen as the radionucleide as it

is easily available and cost effective with a low radiation dose. Moreover, the half life of Tc is only 6h,

which ensures a lower radiation burden when compared to other radio-nuclides like I128 possessing a

longer half life of ~60 days. ITLC analysis revealed that the conjugate 1 was labeled with more than 98%

efficiency. Of note, radiolabeled CNTs were exclusively used for in vivo biodistribution studies,

specifically for quantifying the concentration of free drug as well as CNT-prodrugs in all major

organs/tissues including tumor.

3.2. Physicochemical characterization of MWCNTs

The particle characteristics of acid-oxidized carboxylated MWCNTs, EDBE-MWCNTs, AF-FA-

MWCNTs, AF-FA-MWCNTs (OH) and AF-FA-MTX-MWCNTs have been summarized in Table 1.

Oxidized MWCNTs present an average hydrodynamic size and zeta potential of 173.8±4.2 nm and -

57.5±2.7 mV respectively. Following functionalization with EDBE, the average hydrodynamic size of

CNTs increased to 225.9±3.7 nm with concomitant reversal of surface polarity i.e. the EDBE-MWCNTs

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became positively charged due to the presence of free amine groups on their surface. The conjugation of

FA resulted in further increase of hydrodynamic size and surface polarity reverted to negative, as in the

case of oxidized. CNTs. Accordingly, the hydrodynamic size, PDI and zeta potential of MWCNT-AF-

647-FA-GLY-MTX were analyzed to be 413.1±10.3 nm, 0.411± 0.036 and -24.5± 2.3 mV respectively.

Representative scanning electron micrographs (SEM/TEM) of plain oxidized and FA-MTX-conjugated

MWCNTs compared to aggregated pristine material have been presented in Figure S1 (A) [See

supporting information. All functionalized MWCNTs, irrespective of their surface functionality,

presented well-individualized structure with length ranging between 400-700 µm and diameter 20-60 nm.

Representative TEM image of pristine, 3h-oxidized and FA-MTX conjugated MWCNTs are presented in

Figure S2(B). Consistent with SEM observations, all f-MWCNTs presented an average length of 0.5±0.1

µm. Although acid-oxidation led to significant shortening of CNTs’ length, no visible detrimentation in

structural integrity was evident. We, however, observed some aggregation in the TEM image of our 3h

oxidized MWCNTs, which, possibly, is a consequence of the removal of dispersing phase during sample

preparation and is not representative of the real state of nanotubes in suspension. To further note, all f-

MWCNTs prepared in course of the study presented appreciable dispersibility in aqueous solution and

buffers. The immobilization of various functional molecules on MWCNTs was preliminarily studied

using FTIR and finally authenticated through HRMAS-NMR spectroscopy (See supporting information,

Figure S2-S3 for selected spectral data).

3.3. In vitro stability/ drug release studies

In order to check the stability of the ester linkage between MTX and 1, the release behavior of MTX from

MWCNTs was studied under different conditions: (i) PBS (pH 7.4) (ii) (ii) A549 cell extract iii) rat

plasma (pH 7.4) and (iv) pH 4.5 in presence of lysozymes (to mimic the endosomal conditions). The order

of stability was as follows: PBS > rat plasma >>A549 cell extract > simulated lysosomal fluid (SLF) In

PBS, the release of MTX from MWCNTs was less than 10% even after 48 h of incubation (Figure 3).

Since our formulation was meant for intravenous administration, the release profile of MTX from

MWCNTs was analyzed in serum (rat plasma) as well. As evident from our analysis, approximately 25-

28% of the drug was released from the conjugate over a period of 48h. The degradation may be attributed

to the presence of certain esterases in serum, which is believed to accelerate the cleavage of ester bond.

Moreover, although the major mechanism of drug loading in CNTs is covalent conjugation with the

surface pendant ester groups, some possibility of drug loading through supramolecular π-π stacking

interaction or physical adsorption cannot be completely ruled out. It is possible that drug loaded to CNTs

via relatively weaker interaction may be released in the plasma. Although stability of the conjugate in

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serum is somewhat lower as compared to PBS, the value is still higher when compared with the stability

profile in A549 cell extract or SLF. The release of MTX from MWCNTs (~66% after 48h) was drastically

increased in presence of A549 cell extract. Even higher was the release rate (>85%) under acidic

endosomal conditions. These results suggest that the ester linkage between MTX and MWCNTs are more

stable in serum, as compared to intracellular milieu. In this connection, it may be noted that serum

hydrolyses are more effective on alkyl/ aryl esters with relatively simple chemical structures, particularly,

those containing a lower degree of substitution45-47. Comparatively, cell lysates, tissue homogenates etc.

contain a broad variety of specific as well as nonspecific protease/ esterase, which can act more

efficiently on complex substrates. Although it was not possible to identify the enzymes responsible for the

cleavage of ester bond between MTX and FA-MWCNTs, it is clear that a different classes of enzymes act

on the substrate in plasma and cell lysate. It seems that the presence of substitutents at C-2 and C-3 of the

ester oxygen prevents premature degradation of the ester linkage in serum. The higher stability of the

drug in serum also suggests a greater probability of the prodrug being intact for targeted action. As the

current formulation was meant for active targeting, it is expected that a significant percentage of the

injected dose will accumulate in the target site within 3-4 h of intravenous injection. Within this short

time span, the release of MTX from MWCNTs in plasma is less than 10% so there is minimal chance of

drug loss through chemoenzymatic degradation in plasma. Of note, our results are in line with a

previously reported study on the synthesis and characterization of amino acid ester prodrugs wherein the

authors observed that all prodrugs synthesized in course of the study presented significantly higher

stability in human plasma compared with their stability in Caco-2 homogenates48. These results

strengthen our expectation that the tetrafunctional conjugate developed in course of the study will be

stable in serum but can be converted to the active prodrug immediately after its internalization by target

cells through chemoenzymatic intervention. It was further interesting to note that the amide-linked

conjugate of MTX (2) showed nominal (<15%) release in both plasma and cell lysate. The release rate

was somewhat higher in simulated lysosomal fluid (35-40% after 48h of incubation). However, the value

is still lower when compared to the cleavage profile of 1 in SLF. These results suggest that the ester-

linked conjugate 1 will be more biologically active than its amide linked counterpart (3).

3.4. Stability of the radiolabeled formulation

The in vitro stability of the labeled formulations including 99mTc-AF-FA-MTX-MWCNTs was evaluated

in PBS (pH=7.4) as well as serum. All the formulations showed excellent stability in vitro. The

formulations were approximately 91-94% stable even after 24 h incubation in PBS (Table S1, Supporting

information). Similar trend was observed in serum (data not shown). As for the in vivo stability is

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concerned, approximately 85-87% of all MWCNT-conjugates remained labeled in vivo, even after 24 h

of injection. Labeling efficiency of free MTX was determined to be approximately 82% after 24 h,

implying that both radiolabeled CNTs and free MTX are stable in the physiological milieu.

3.5. Ability of AF-FA-MWCNTs to target and destroy FR (+ve) cancer cells

The ability of 1 to target FR (+ ve) cancer cells was evaluated by confocal microscopy. The results clearly

indicated that 1 was selectively internalized by FR (+ve) A549 and MCF cells (Figure 4A, 4B, upper

panel) when compared to FR (-ve) Neuro 2A cells presenting nominal internalization (data not shown). It

was interesting to observe that the uptake of the conjugate in both cell lines was significantly inhibited in

the presence of free FA (Figure 4A, 4B, lower panel) suggesting the involvement of FR in the cellular

uptake of nanotubes. The therapeutic conjugate concentrated mainly in the cellular cytoplasm leaving a

clear zone of nucleus. These results indicated that AF-FA-MWCNTs cannot transport through the nuclear

envelope. It is worthy to mention that nuclear pore on the nuclear membrane allows mRNA and tRNA to

cross the nuclear envelope, and as apparent from various reports, the diameter of these pores have been

estimated to be around 10–20 nm. As evident from SEM imaging, diameter of the prodrug-conjugate

ranged from 40-50 nm, which was too large to traverse the nuclear pore.

The ability of FA-MTX cotethered MWCNTs to destroy cancer cells was studied by MTT assay. To

prove the FR-mediated targeting of CNTs, FR (+ve) A549 and MCF 7 cell lines were incubated with 1.

MTX deprived EDBE-MWCNTs, AF-FA-MWCNTs were taken as control. To elucidate the role of

surface functional molecules and associated linkers on the targeting and therapeutic efficacy of the

synthesized prodrug, two additional formulations viz. conjugates 2 and 3 were tested. While MTX-

deprived MWCNTs led to negligible reduction in cellular viability (IC-50 >> 10 µg/ml), both free MTX,

1 and 3 exhibited dose dependent reduction in cellular viability (Figure S4). The IC-50 values of free

MTX/ CNT-MTX conjugates in A549 and MCF 7 cells (normalized to MTX concentration) have been

furnished in Table 2. In either cell lines, the conjugate 1 exhibited the highest toxicity, followed by 3 and

free MTX. Two possible mechanisms might have been responsible for the increased cytotoxicity of 1 as

compared to free MTX and 3: (i) increased cellular uptake mediated by targeted interaction with folate

binding protein (FBP) and/ or reduced folate carrier (RFC); (ii) increased affinity/ intrinsic activity of

CNT-conjugated MTX to dihydrofolate reductase (DHFR) enzyme. To understand the contribution of

these two mechanisms, A549 and MCF 7 cells were preincubated with excess FA (50 µg/ml) for 1h and

exposed to conjugates 1 and 3. It was interesting to observe that cytotoxicity of 1 in both cell lines was

significantly suppressed in presence of free FA, suggesting the involvement of an FR mediated pathway

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responsible for the internalization of 1 by its molecular target. Remarkably, similar reduction in

cytotoxicity was not observed in case of 7 indicating that presence of MTX moieties on the surface of

CNTs do not facilitate binding with FRs (data not shown). The increased anticancer activity of 1

compared to 3 could have been attributed to an increased cellular uptake via FR mediated endocytosis.

However, both 1 and 3 showed appreciable interanalization by A549 and MCF cells (Figure S5)

suggesting that surface functionality of CNTs have nominal influence on the extent of cellular uptake.

Since MTX has a poor cellular uptake and low target specificity, the higher anticancer activity of 1 and 3

over free MTX suggests that intracellular concentration of MTX delivered through MWCNTs is higher

than that of free MTX. Unfortunately, the same explanation could not be extrapolated for justifying the

higher anticancer activity of 1 over 3. Furthermore, if increased cellular uptake is regarded as the sole

driving force behind increased anticancer activity of CNT-MTX conjugates, the efficacy of the amide-

linked conjugate 2 would have been comparable with 1 and 3. Paradoxically, the conjugate 2 exhibited

nominal cytotoxicity even at the highest concentration of incubation (100µg/ml). In order to verify

whether any difference exists in enzyme inhibiting activities of CNT-bound MTX and free MTX, DHFR

activity assay was also performed. Interestingly, none of the CNT-MTX conjugates showed any inhibition

of DHFR activity in A549/ MCF 7 cell lysates (data not shown). To rationalize the lack of DHFR

inhibiting activity of 1/3, docking studies were performed on human DHFR (PDB ID=1U72). The

docking of free MTX and 1 with DHFR has been presented in Figure 5 (a, b). As evident from docking

studies, CNT-bound MTX was unable to reach the binding pocket of DHFR. In an attempt to account for

the observed inactiveness of MTX in bound form, ligand similarity analysis of DHFR bound free MTX

and CNT-bound MTX was performed. Figure 5 (c) presents the docking conformation of free MTX. The

blue cloud in Figure 5(d) represents the DHFR bound conformation of free MTX. The snapshot elucidates

that CNT conjugated MTX cannot achieve the desired conformation necessary to exert inhibitory action

on DHFR and supports our docking results. It seems that the steric hindrance posed by multiple functional

guests on the surface of 1/ 3 does not allow CNTs to achieve the favorable conformation essential for

showing DHFR activity.

These findings were also consistent with the results of in vitro cellular uptake and cytotoxicity studies

according to which conjugation of MTX with FA-MWCNTs hardly endowed any additional targeting

effect to the nanotubes. Thus, although cellular uptake of MTX is higher when delivered through CNTs,

the drug fails to exert the desired therapeutic activity unless it is cleaved from the nanotube surface. This

also explains the relatively higher activity of MTX when linked to CNTs via ester linkage as compared to

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amide linkages with a slower rate of hydrolysis. The result was supported by in vitro stability studies

under abiotic conditions in the presence of simulated lysosomal fluid, cell extracts and serum [Figure 3].

The results can be better explained if we look into the intracellular trafficking of 1 and 3. Our cell uptake

studies revealed that the conjugate 1 was mainly confined to cytoplasm leaving a clear nuclear zone. To

further elucidate whether prodrug conjugates, synthesized in course of our study, can accumulate in thecal

organelles, lysosomal colocalization experiments were performed. The colocalization of AF-488 labeled

MWCNTs in NR stained lysosomes was studied using confocal microscopy. Colocalization in the entire

field of view was determined through scattered plot analysis. The extent of colocalization between AF-

488 labeled CNTs and an NR stained lysosomes was measured in terms of Pearson’s correlation

coefficient (r); as a thumb rule, a colocalization coefficient close to or greater than 0.5 (r ≥0.5) was

considered as indicator of good colocalization. As evident from Figure 6(A), the conjugate 1 showed

appreciable compartmentalization in lysosomes (r>0.6) whereas the prodrug 3 was mainly restricted to

cytoplasm [Figure 6(b)] without any vesicular accumulation (r<0.3). Consequently, the higher anticancer

activity of 1 over 3 can be explained on the basis of their differential uptake mechanism and

transmembranal pathways. While FA moiety in 1 facilitated endolysosomal trafficking of MWCNTs, the

conjugate 3 was mainly restricted to cytoplasm. Once the folate-targeted conjugate (1) binds with its

cognate receptor, the invaginating plasma membrane envelops the ligand-receptor complex, forming

endosomes49. These endosomes are trafficked intracellularly to lysosomes wherein the ester bond between

MTX and CNTs is easily cleaved in presence of cellular esterases at pH 7.4 or lysozymes under low pH

conditions, thereby increasing the intracellular bioavailability of free MTX. A similar uptake or cleavage

mechanism is not operative in case of 3 as internalization of the conjugate is independent of FBP/ RFC.

The results of in vitro stability studies suggest that the transformation of the prodrug to its active form

will be faster in lysosomes as compared to cytosol. This explains, in part, the lower antiproliferative

activity of 3 as compared to 1. Although the amide-linked conjugate 2 showed some localization in the

lysosomal compartment due to the presence of FA (data not shown) as the targeting moiety, the conjugate

presented nominal toxicity (IC-50 >> 10 µg/ml). Such a behavior may be attributed to an exceptionally

slower rate of hydrolysis of 2 in the intracellular milieu.

3.7. In vivo evaluation of AF-FA-MWCNTs

3.7.1. Organ distribution Study

In order to evaluate the potential significance of 1 as tumor-targeted theranostic prodrug, bio-distribution

of 99mTc-AF-FA-MTX-MWCNTs was evaluated in Ehlrich Ascites Tumor (EAT) bearing mice model, a

well-established xenograft model for FR over-expressing tumors. The conjugates 3, 4 and free MTX were

used as control. The percentage injected dose per gram (% ID/g) of tissue in different organs at different

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time points (0.5, 2, 4 and 24h) has been presented in Table 3 (a-d). As evident from organ distribution

data, both free drug and radio-labeled MWCNTs exhibited nominal accumulation in heart, stomach and

intestine. Compared to free MTX, all functionalized MWCNTs including the PEGylated control presented

a high initial accumulation of CNTs in the organs of mononuclear phagocytic system (MPS). These

results are in line with the observations of Qu et al. according to which both agglomerated and well

suspended CNTs are taken up by liver, spleen and lungs after i.v. injection 50. While MWCNTs with

higher degree of agglomeration are retained in lungs and later in the liver for months, the well-dispersed

ones are easily eliminated from the body via excretion. In the present case, the % injected dose (ID)/g in

the MPS organs decreased as function of time, revalidating that well-functionalized and individualized

CNTs show minimum propensity towards bioaccumulation and are ultimately, eliminated via urinary

excretion or biliary pathway in the faeces. It was further interesting to observe that %ID/g of 1 (AF-FA-

MTX-MWCNTs) and 4 (PEGylated MWCNTs) in liver and spleen was almost comparable implying that

covalent linking of FA and other functional bioactives through a short PEG-like spacer (EDBE) enables

the nanotubes to avoid permanent sequestration by the MPS organs. Compared to 1, 3 and 4, free MTX

showed more rapid clearance from blood circulation through urinary excretion, as evident from the high

%ID/g of 99mTc-MTX in kidney after 0.5 and 2h of i.v. administration. Consequently, accumulation of

free drug at the tumor site after 4h of injection was even less than 0.1%ID/g. Conversely, all MWCNT

conjugates viz. 1, 3 and 4 showed appreciable accumulation in the tumor site [Table 3 (b-d)]. The tumor

to muscle ratio for all the conjugates increased as function of time up to 4 h post-administration. The

formulations were able to retain in these sites even after 24 h of administration. After 24 h, the tumor to

muscle ratio for free MTX, 3, 4 and 1 were calculated to be 1.5, 3.33 and 26.72 respectively, implying

that tumor-specific accumulation of FA-MTX cotethered MWCNTs is around 19.14 and 8.62 times

higher as compared to free MTX and FA non-targeted conjugate 3. Appreciable accumulation was

observed for PEGylated MWCNTs which may be a consequence of passive tumor targeting via enhanced

permeability and retention (EPR) effect. Of note, the bio-distribution profile of 1 and AF-FA-MWCNTs

were almost comparable (data not shown), indicating that covalent conjugation of MTX with MWCNTs

hardly endows any additional targeting effect to the carrier system.

3.7.2. Tumor growth inhibition study

In vivo tumor growth inhibition study was carried out in DMBA induced Sprague Dawley female rats,

administered i.v. twice with 5 mg/kg of free MTX and f-MWCNTs in equivalent concentration of free

MTX at weekly intervals. Figure 7 presents the tumor-growth inhibition profile of rats treated with

various f-MWCNT preparations. A single i.v. administration of 1and 3, on average, led to 38.37 and

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25.37 % reduction in tumor burden respectively within 24 h of administration. Despite an initial decrease

in tumor burden, both free drug and CNT-treated groups showed some proclivity towards tumor

recurrence from the 4th day of treatment. Although tumor burdens of mice treated with 1 and 3 were

considerably lower than either free drug or untreated control, a second dosing was administered on the 8th

day to arrest further recurrence. Amongst all the treated groups, the conjugate 1 exhibited the highest

antitumor efficacy. Reduction in tumor volume for rats treated with 1 for 15 days was approximately 9.2,

9.1, 2.6, 4.1 and 2.1 times higher as compared to rats exposed to saline, AF-FA-MWCNTs, free MTX, 2

and 3 respectively. Remarkably, 2 out of 4 rats treated with 1 showed more than 80% tumor regression on

the very second day of treatment and tumor completely disappeared within a week with no recurrence

during the entire treatment course (See supporting information, Figure S6). The high anticancer activity of

1 might be attributed to high tumor-specific accumulation of f-MWCNTs, facilitated through (i) FBP/

RFC; (ii) intrinsic cell penetrability of CNTs and (iii) easy cleavability of MTX from CNTs through

chemoenzymatic hydrolysis of ester bond. It was also interesting to note that throughout the treatment

course, tumor-growth inhibitory effect of 3 was higher than 2, revalidating that cleavability of bonds

between drug and nanotube is a critical factor to determine their future application in vivo.

3.7.3. Toxicity study

The toxicity of free MTX/ MTX-CNT conjugates was evaluated in mouse model using the same dose

used in efficacy studies. As evident from biodistribution studies [Table 3 (a-d)], both free MTX, 1 and 3

showed significant accumulations in liver. The organ indices of mice treated with free MTX as well as

functionalized MWCNTs have been presented in Figure S7(a). While MWCNT-treated groups showed

insignificant differences from control, liver index of mice exposed to free MTX was less than the control,

indicating the possibility of free drug induced hepatotoxicity. The AST and ALT levels in mouse blood at

15 days post-exposure with free MTX/ various CNT formulations have been presented in Figure S7 (b).

In either case, for f-MWCNT treated groups, no significant change with respect to control was observed,

suggesting that well-functionalized MWCNTs induce minimal hepatotoxicity in mice. Notably, the AST

level of mice (29.15 ± 2.35) treated with free drug was significantly higher than that of control and rather

close to the upper limit of normal (ULN ~ 30 IU/L). Likewise, the LDH level of animal groups treated

with free MTX was considerably higher than the control while for the f-MWCNT treated groups, the

difference between the control and treatment groups were almost mitigated. These results are not against

streamline because MTX therapy is often associated with elevation of AST/ ALT. In low dose, MTX

therapy may lead to fibrosis/cirrhosis of liver, which, in certain instances even, mature to hepatocellular

carcinoma. Similarly, high-dose MTX therapy often leads to distorted liver function tests51, 52. Herein,

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MTX treatment led to decreased liver index as well as increased AST and LDH levels, which, however

was not observed in case of any of the CNT-MTX conjugates. These observations implied that

conjugation of MTX with well functionalized MWCNTs is an effective way to alleviate the drug-

associated hepatotoxicity. Figure S7(c-d) presents the MDA levels and superoxide dismutase (SOD)

activity in liver, following 15 days post-administration of free MTX/ CNT-conjugates in mice. Of note,

MDA level and SOD activity of all MWCNT treated groups showed insignificant differences from the

control, signifying that CNT-conjugates, developed in course of the study, induce minimal oxidative

stresses. In our previous reports, we have already shown that surface functionality had nominal influence

on CNT-induced oxidative stress. CNT-mediated reactive oxygen species (ROS) generation is critically

dependent on surface hydrophobicity and metal impurities associated with the pristine material39, 53. In

the present study, all functional molecules were attached to CNTs via a hydrophilic PEG-like spacer

(EDBE), which rendered the overall carrier system hydrophilic. Furthermore, as determined from

electron-dispersive X-ray (EDAX) analysis, all MWCNT preparations were highly pure containing

negligible amount of metallic impurities (data not shown), which were too low to initiate any ROS

production inside the cells. To further examine whether f-MWCNTs induce any cardiotoxicity in mice,

the various markers of cardiotoxicity including heart index, AST/ALT, CK-MB, LDH, MDA and SOD

were analyzed. As shown in Figure S7 (b, c), AST and LDH levels of free MTX treated group were

significantly higher than the control and the values marginally exceeded the ULN. Otherwise, mice

treated with either MTX or functionalized MWCNTs showed insignificant difference from the control

(data not shown) suggesting that our formulations do not induce any obvious cardiotoxicity in mice. The

blood urea nitrogen (BUN) levels of free MTX as well as f-MWCNT treated groups after 15 days post-

injection have been presented in Figure S7 (e). The results corroborate that neither free MTX nor

functionalized MWCNTs induce any nephrotoxicity in mice.

4. Conclusion.

In conclusion, a novel, theranostic prodrug based on FA-MTX cotethered MWCNTs has been synthesized

by concomitant decoration of acid-oxidized MWCNTs with four different functional moieties: a

fluorochrome (viz. Af-488/647), a targeting ligand (FA), a chemotherapeutic agent (MTX) and a radio-

tracer (99mTc). In course of extensive in vitro, in silico and in vivo studies, we established that the delivery

property of MWCNTs, high tumor binding avidity of FA, optical detectability of AF fluorochromes and

radio-traceability of 99mTc could be successfully cocktailed on a single platform to augment the

therapeutic efficacy of MTX against FR over-expressing cancer cells while allowing a real-time

monitoring of drug delivery and treatment response through combined optical and scintigraphic imaging.

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Our results indicated that internalization, subcellular translocation, cytotoxic responses, bio-distribution

and therapeutic efficacy of any chemotherapeutics-integrated CNT platform critically depends on its

surface chemistry and associated linkers. Thus, covalent conjugation of MTX through a serum stable yet

intracellularly hydrolysable ester linkage not only augmented the site-specificity and antitumor efficacy of

the drug but also alleviated its deleterious effects against normal cells/tissues. Although further studies are

necessary to determine the long term fate of our functionalized MWCNTs, the multifunctional CNTs

developed in course of the study can be effectively used for expanding the theranostic window for a broad

spectrum of anticancer agents including MTX.

Acknowledgements. The authors are thankful to Indian Council of Medical Research (ICMR, Grant no.

No: 35/28/2010/-BMS) and Department of Science & Technology (DST), Government of India, New

Delhi, for financial support. Director NIPER and Director INMAS are duly acknowledged for providing

the necessary infrastructure and facilities. Thanks are due to Mrs. Bhupindar Kaur, Dept. of anatomy, PGI

Chandigarh and Dr. Ravi S. Amarpati, SAIF, Central Drug Research institute (CDRI, Lucknow) for

assistance with flow cytometry and HRMAS-NMR analysis. Technical assistance of Mr. Dinesh Singh

Chauhan and Mr. Rahul Mahajan is also acknowledged.

Supporting Information Available. This information is available free of charge via the Internet at

http://pubs.acs.org/.

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Figures and Tables

Figure 1. A schematic representation illustrating the multifunctionalization of MWCNTs. AF and FA were coupled to MWCNTs via relatively stable amide linkage whereas MTX was conjugated through an intracellularly hydrolyzable ester linkage. 99mTc was coordinated with MWCNTs through negatively charged hydroxyl (–O-)/ carboxyl (-COO-) groups on the surface of MWCNTs.

H2NO O NH2

(i) H2SO4/HNO3

(ii) SOCl2,

Pristine-MWCNTs

reflux, 24 h

excess, 12h

O O

OO

O O

OO

HN HN HNHN

NH NHNH

NH

[O]

EDBE

NH2 NH2NH2

NH2

H2NH2N H2N

H2N

NN

N

HNO

NH

O

NH2

COOH

N OH

O

N

O

O

FA-NHS ester

rt, stirring, 18 h, dark

O

OO

O O

OO

HN HNHN

NH NHNH

NH

NH NH2

NHNH2

HN H2NHN

AF-488/647)

[50:1]

OO

OO

AF

FA FA

FA

FA

AF

NN

N

NO

NH

O

NH2

COOH

N NH2

OH

EDC, DMSOPyridine

Activated MTX

48 h, stirring, dark

Amine-MWCNTs

O

OH

ETOH, Et3Hrt, stirring, 12 h, dark

Glycidol

O

OO

OO

OO

HN HNHN

HN

NH NHNH

HN

NHN

HN N NH

O

O

O

O

AF

FAFA

FA

AFN

AF

OH

HO

O

O

OH

OH

O

OOH

HOOOO

XTMOXTM O MTX

O

MTX

OMTX

OMTX

Technitium-99mO

O

HN

NH

N

NH

OFA

AF-647

OH

OH

O

O

OMTX

O

MTX

Tc (99m)

(1)

AF-FA-MWCNTs

AF-FA-MTX-MWCNTs

O

OO

O O

OO

HN HNHN

HN

NHNH

NHHN

NHN

HN NNH

O

O

OO

AF

FAFA

FA

FA N

AF

OH

HO

HO

HO

OH

OH

OH

OHOH HO

OHOH

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Figure 2. Chemical structures of MWCNT-conjugates 1-4

O

OO

O O

OO

HN HNHN

HN

NH NH NHHN

NH N

HN N NH

O

O

O

O

AF

FA FA

FA

FAN

AF

OHHO

O

O

OH

OH

O

OOHHO

OO

MTXOXTM

O MTX

O

MTX

OMTX

OMTX

(1)

O

O

O

O

O

O

O

NH

NH

NH

HN

HN

HN

HN

HN

NH2

NH

NH

NH

H2N

NH

O

O

O

O

FA

XTM

FA

MTX

FA

AF

(2)

O

AF

O

OO

O O

OO

H2N HNHN

HN

NH NH NHH2N

NH2 N

N NH2

AF

N

AF

OH

HO

O

O

OH

OH

O

OOHHO

OOO

MTXOMTX

O MTX

O

MTX

OMTX

OMTX

(3)

OH

O

O

O

HO

O

O

O

O

O

O

OHO

O

O

O

OCH3

O

O

OCH3

n

n

(4)

(4)

m-PEG m-PEG

m-PEG

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Figure 3. In vitro stability studies: The ester linked conjugate, 1(a) shows faster hydrolysis in the intracellular milieu as compared to its amide linked counterpart (b).

(A)

0 10 20 30 40 500

20

40

60

80

100

120

140

160

C

um

ula

tive M

TX

Rele

ase (%

) SLF

A549 cell lysate

Rat Plasma

PBS

Time (h)0 10 20 30 40 500

20

40

60

80

100

120

140

160

Cum

ula

tive M

TX

Rele

ase (%

)

Time (h)

SLF

A549 cell lysate

Rat Plasma

PBS

(a) (b)

50 µm50 µm 50 µm

50 µm50 µm50 µm

(a)

(b)

(i) (ii) (iii)

(i) (ii) (iii)

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(B)

Figure 4. In vitro cell uptake studies: FR (+ve) A549 (A) and MCF 7 (B) cells were incubated with AF-647-FA-MTX-MWCNTs (1) in absence (upper panel) and presence (lower panel) of free FA. For each incubation type, (i), (ii) and (iii) represents DAPI fluorescence (blue) AF 647 fluorescence (red) and merged fluorescence of DAPI and AF 647 respectively.

Figure 5.In silico docking and ligand similarity analysis of free MTX and CNT-MTX conjugates:

(a, b) Docking of free MTX and FA-MTX-MWCNT conjugate with DHFR (PDB ID=1U72) respectively; (c) Docking conformation of MTX; (d) Ligand similarity analysis of CNT bound MTX with DHFR bound conformation of free MTX.

50 µm50 µm50 µm

50 µm50 µm 50 µm

(a)

(b)

(i) (ii) (iii)

(i) (ii) (iii)

(a) (b) (c) (d)

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Figure 6. Intracellular trafficking of CNT-MTX conjugates: Confocal microscopy images of A549 cells incubated with (A) 1 and (B) 3. For each incubation type, (i), (ii) and (iii) represents fluorescence images of NR stained lysosomes (red fluorescence), AF-488 labelled multifunctional MWCNTs (green fluorescence) and merged fluorescence image of NR and AF-488 respectively; (iv) represents scatter plot.

(i) (ii)

r=0.64 (iv)(iii)

(A)

r=0.28 (iv)

(ii)

(iii)

(i)

(B)

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Figure 7.Pharmacodynamic assessment in rats: Tumor growth inhibition properties of free MTX and CNT-MTX conjugates (1-3) in chemically tumor-induced SD rats.

0 2 4 6 8 10 12 14 160

100

200

300

400

Control

FA-CNTs

MTX

2

3

1

Tum

or

Volu

me (%

)

Time (Day)

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Tables

Table 1: Particle characteristics of functionalized MWCNTs

Conjugate studied Hydrodynamic size

Zeta Potential

PDI Particle size (Length) 1,2

- nm mV nm Oxidized MWCNTs 173.8±4.2 0.421±0.052 -57.5±2.7 400-700 EDBE-MWCNTs 230.9±3.7 0.437±0.034 +9.2±0.6 400-700 FA-MWCNTs 361.3±8.7 0.280±0.021 -21.6±4 400-700 FA-MTX-MWXNTs 413.1±10.3 0.411±0.036 -24.5± 2.3 400-700

1Diameter of f-MWCNTs ranged on average from 30-50 nm as observed under a scanning electron microscope (SEM, Model S30400); 2Representative SEM images of selected f-MWCNTs are provided as additional supplementary figure, Figure S1. Table 2: IC-50 values of free MTX and CNT-MTX conjugates in A549 and MCF 7 cell lines

Sample examined IC-50 (µg/ml), A549 cells IC-50 (µg/ml), MCF 7 cells Free MTX 7.26 7.36

3 5.32 5.19 1 2.13 1.95

Table 3 (a) In vivo bio-distribution profile of free 99mTc-MTX

Organ or tissue % ID /g recovered after 0.5h 2h 4h 24h

Blood 13.29 ±0.89 7.27±0.98 4.12±0.49 0.27±0.9 Heart 0.10±0.02 0.10±0.02 0.06±0.01 0.04±0.01 Lungs 3.65±0.12 5.26±0.34 2.10±0.13 0.16±0.02 Liver 10.61±1.36 15.81±1.12 10.35±0.87 0.48±0.04 Spleen 8.36±0.62 11.25±0.75 8.83±.65 0.51±0.03 Kidney 21.25±1.37 25.15±2.65 29.64±2.47 1.30±0.17 Stomach 0.06±0.01 0.08±0.02 0.07±0.01 0.05±0.01 Intestines 0.07±0.01 0.10±0.02 0.08±0.01 0.07±0.01 Tumor 0.21±0.05 0.23±0.02 0.26±0.05 0.06±0.02 Muscle 0.17±0.03 0.14±0.06 0.09±0.02 0.04±0.01 Tumor: Muscle 1.23 1.64 2.88 1.5

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Table 3 (b) In vivo bio-distribution profile of free 99mTc-AF-MTX-MWCNT (3)

Table 3 (c) In vivo bio-distribution profile of free 99mTc-MWCNT-PEG (OCH3) (4)

Table 3 (d) In vivo bio-distribution profile of free 99mTc-MTX-FA-AF-MWCNTs (1)


Blood 0.74±0.03 0.62±0.03 0.46±0.02 0.12±0.02 Heart 0.23+0.02 0.17+0.04 0.15+0.06 0.06±0.01 Lungs 24.65 ± 0.55 19.2 ±0.4 15.8 ±0.7 5.3 ±0.4 Liver 28.46 ±1.38 16.2 ±0.7 14.26 ±0.03 8.35±0.65 Spleen 12.6 ± 0.2 6.71 ±0.03 5.65 ±0.45 4.35 ±0.15 Kidney 1.87+0.13 0.92 ±0.04 0.63 ±0.05 0.49 ±0.05 Stomach 0.53 ±0.03 0.49 ±0.01 0.42 ±0.02 0.26 ±0.02 Intestines 0.66 ±0.05 0.54 ±0.02 0.32 ±0.02 0.11 ±0.01 Tumor 1.68 ±0.12 1.92 ±0.18 2.02 ±0.12 1.14 ±0.13 Muscle 0.44 ±0.08 0.36±0.07 0.17 ±0.01 0.34 ±0.08 Tumor: Muscle 3.81 5.3 5.94 3.33

Organ or tissue

% ID /g recovered after 0.5h 2h 4h 24h

Blood 0.83±0.03 0.62±0.03 0.49±0.02 0.21±0.02 Heart 0.23+0.02 0.18+0.01 0.13+0.02 0.05±0.01 Lungs 22.65 ± 0.55 14.2 ±0.4 12.8 ±0.7 4.3 ±0.4 Liver 26.13 ±1.48 12.5 ±1.7 10.21 ±1.12 7.16 ±0.69

Spleen 11.8 ± 0.2 5.91 ±0.03 4.45 ±0.48 3.15 ±0.15 Kidney 1.69+0.18 0.92 ±0.12 0.65 ±0.05 0.47 ±0.05

Stomach 0.52 ±0.03 0.45 ±0.11 0.43 ±0.08 0.26 ±0.02 Intestines 0.65 ±0.05 0.42 ±0.02 0.31 ±0.02 0.14 ±0.01

Tumor 1.92 ±0.16 2.08±0.005 2.21 ±0.12 1.16 ±0.03 Muscle 0.38 ±0.08 0.24 ±0.02 0.34 ±0.01 0.30 ±0.02

Tumor: Muscle 4.52 8.66 7.11 3.86


Blood 0.61±0.03 0.46±0.03 0.34±0.05 0.285±0.025 Heart 0.19±0.03 0.12±0.01 0.09±0.004 0.03±0.02 Lungs 23.3 ± 0.4 1.57±0.11 1.275±0.045 0.945±0.035 Liver 26.95±0.35 9.165±0.465 7.831±0.241 5.3 ±0.4 Spleen 12.13 ± 0.25 3.66±0.37 2.84±0.075 0.78±0.03 Kidney 1.54±0.07 0.63±0.06 0.465±0.015 0.12±0.01 Stomach 0.46±0.06 0.36±0.03 0.29±0.07 0.055±0.005 Intestines 0.23±0.02 0.17±0.02 0.133±0.05 0.04±0.01 Tumor 3.28±0.02 4.44±0.005 4.46±0.121 2.56±0.26 Muscle 0.60±0.09 0.252±0.005 0.22±0.02 0.096±0.005 Tumor: Muscle 5.46 17.61 22.09 26.66

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34

Table of Contents Use Only

Augmented Anticancer Activity of a Targeted, Intracellularly Activatable, Theranostic

Nanomedicine based on Fluorescent and Radiolabeled, Methotrexate-Folic acid-

Multiwalled Carbon Nanotube Conjugate

Manasmita Das, Satyajit R. Datir, Raman Preet Singh, Sanyog Jain*

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476x688mm (96 x 96 DPI)

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476x549mm (96 x 96 DPI)

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476x198mm (96 x 96 DPI)

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476x320mm (96 x 96 DPI)

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476x323mm (96 x 96 DPI)

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1375x325mm (96 x 96 DPI)

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317x300mm (96 x 96 DPI)

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370x342mm (96 x 96 DPI)

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317x267mm (96 x 96 DPI)

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555x406mm (96 x 96 DPI)

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Documents

Augmented Anticancer Activity of a Targeted, Intracellularly Activatable, Theranostic Nanomedicine Based on Fluorescent and Radiolabeled, Methotrexate-Folic Acid-Multiwalled Carbon