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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, and Sanyog Jain Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp300701e • Publication Date (Web): 17 May 2013 Downloaded from http://pubs.acs.org on May 19, 2013

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Molecular Pharmaceutics

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)

1

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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 AFFA-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 radiotraceability of

99m

Tc 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 realtime 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. trifunctionalized 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 SWCNTmediated 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 3



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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 (FAMTX) 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 biodistribution

of

the

nanovector

through

combined

optical

imaging

Multifunctionalization of MWCNTs with FA, MTX, AF-fluorochrome and

and

radioscintigraphy.

99m

Tc 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,

99m

Tc 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],

dihydrochloride (DAPI)

4′,6-diamidino-2-phenylindole

and 7,12 Dimethylbenz [α]anthracene (≥ 95% pure) were purchased from 4


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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 5



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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, recentrifuged 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 Nhydroxy 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 method using stannous chloride (SnCl2) as the reducing agent

26-28

99m

Tc by direct labeling

. 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 6


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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 MWCNTMTX conjugates in which MTX was conjugated to CNTs via ester bond (3). For synthesis of amidelinked 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 fCNT 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 7



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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-MTXMWCNTs 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 0.1100µg/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 9



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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,

99m

Tc-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,

99m

Tc-AF-FA-MTX-MWCNTs and all control formulations was studied

in female Sprague Dawley (SD) rats. Tumors were chemically induced in animals using 7, 12dimethylbenz[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, MWCNTAF-647-FA-GLY-MTX, MWCNT-AF-647-FA-GLY, MWCNT-AF-647-FA and MWCNT-EDBE-AF647 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 singlewalled 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 11



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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/ antiinflammatory 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 serumstable 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 C2/ 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 12


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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

99m

Tc. As described in our earlier reports,

99m

Tc 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-FAMWCNTs, 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 13



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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-AF647-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 fMWCNTs 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 2528% 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 14


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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 (> 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 16


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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 amidelinked 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

17



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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 AF488 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> 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

99m

Tc-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 18


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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-FAMTX-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

99m

Tc-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 19



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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, 20


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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 drugassociated 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 postinjection 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 radiotracer (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

99m

Tc 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. 21



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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/.

References 1. Madani, S. Y.; Naderi, N.; Dissanayake, O.; Tan, A.; Seifalian, A. M. A new era of cancer treatment: carbon nanotubes as drug delivery tools. International Journal of Nanomedicine 2011, 6, 2963. 2. Chakrabarti, A.; Patel, H.; Price, J.; Maguire, J. A.; Hosmane, N. S. Carbon Nanotubes in Cancer Therapy, Including Boron Neutron Capture Therapy (BNCT). Nanotechnologies for the Life Sciences 2011. 3. Bianco, A.; Kostarelos, K.; Prato, M. Opportunities and challenges of carbon-based nanomaterials for cancer therapy. Expert Opinion on Drug Delivery 2008, 5, (3), 331-342. 4. Thakare, V. S.; Das, M.; Jain, A. K.; Patil, S.; Jain, S. Carbon nanotubes in cancer theragnosis. Nanomedicine 5, (8), 1277-1301. 5. Pastorin, G. Crucial functionalizations of carbon nanotubes for improved drug delivery: a valuable option? Pharmaceutical research 2009, 26, (4), 746-769. 6. Dumortier, H.; Lacotte, S.; Pastorin, G.; Marega, R.; Wu, W.; Bonifazi, D.; Briand, J.-P.; Prato, M.; Muller, S.; Bianco, A. Functionalized carbon nanotubes are non-cytotoxic and preserve the functionality of primary immune cells. Nano letters 2006, 6, (7), 1522-1528. 7. Dhar, S.; Liu, Z.; Thomale, J.; Dai, H.; Lippard, S. J. Targeted single-wall carbon nanotubemediated Pt (IV) prodrug delivery using folate as a homing device. Journal of the American Chemical Society 2008, 130, (34), 11467-11476.

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26. Jain, S.; Mathur, R.; Das, M.; Swarnakar, N. K.; K., M. A. Synthesis, pharmacoscintigraphic evaluation and antitumor efficacy of methotrexate loaded, folate conjugated, stealth albumin nanoparticles. Nanomedicine 2011, 6, 1733-1754. 27. Yadav, A. K.; Mishra, P.; Mishra, A. K.; Jain, S.; Agrawal, G. P. Development and characterization of hyaluronic acid-anchored PLGA nanoparticulate carriers of doxorubicin. Nanomedicine: Nanotechnology, Biology and Medicine 2007, 3, (4), 246-257. 28. Pathak, A.; Kumar, P.; Chuttani, K.; Jain, S.; Mishra, A. K.; Vyas, S. P.; Gupta, K. C. Gene Expression, Biodistribution, and Pharmacoscintigraphic Evaluation of Chondroitin Sulfate- PEI Nanoconstructs Mediated Tumor Gene Therapy. ACS nano 2009, 3, (6), 1493-1505. 29. Das, M.; Bandyopadhyay, D.; Mishra, D.; Datir, S.; Dhak, P.; Jain, S.; Maiti, T. K.; Basak, A.; Pramanik, P. Clickable , Trifunctional Magnetite Nanoparticles and their Chemoselective Biofunctionalization: From a Smart Surface to a Smarter Nanoweapon for Cancer Theranostics. Bioconjugate Chemistry. 30. Santra, S.; Kaittanis, C.; Grimm, J.; Perez, J. M. Drug/Dye Loaded, Multifunctional Iron Oxide Nanoparticles for Combined Targeted Cancer Therapy and Dual Optical/Magnetic Resonance Imaging. Small 2009, 5, (16), 1862-1868. 31. Asati, A.; Santra, S.; Kaittanis, C.; Nath, S.; Perez, J. M. Oxidase-Like Activity of Polymerâ€ Coated Cerium Oxide Nanoparticles. Angewandte Chemie International Edition 2009, 48, (13), 2308-2312. 32. Santra, S.; Kaittanis, C.; Perez, J. M. Cytochrome c encapsulating theranostic nanoparticles: a novel bifunctional system for targeted delivery of therapeutic membrane-impermeable proteins to tumors and imaging of cancer therapy. Molecular Pharmaceutics 7, (4), 1209-1222. 33. Santra, S.; Kaittanis, C.; Santiesteban, O. J.; Perez, J. M. Cell-Specific, Activatable and Theranostic Prodrug for Dual Targeted Cancer Imaging and Therapy. Journal of the American Chemical Society. 34. Santra, S.; Perez, J. M. Selective N-Alkylation of Î²-Alanine Facilitates the Synthesis of a Poly (amino acid)-Based Theranostic Nanoagent. Biomacromolecules. 35. Kaittanis, C.; Santra, S.; Santiesteban, O. J.; Henderson, T. J.; Perez, J. M. The Assembly State between Magnetic Nanosensors and Their Targets Orchestrates Their Magnetic Relaxation Response. Journal of the American Chemical Society. 36. Kaittanis, C.; Santra, S.; Perez, J. M. Role of nanoparticle valency in the nondestructive magnetic-relaxation-mediated detection and magnetic isolation of cells in complex media. Journal of the American Chemical Society 2009, 131, (35), 12780-12791. 37. Swarnakar, N. K.; Jain, A. K.; Singh, R. P.; Godugu, C.; Das, M.; Jain, S. Oral bioavailability, therapeutic efficacy and reactive oxygen species scavenging properties of coenzyme Q10-loaded polymeric nanoparticles. Biomaterials 2011, 32, 6860–6874. 38. Jain, A. K.; Swarnakar, N. K.; Das, M.; Godugu, C.; Singh, R. P.; Rao, P. R.; Jain, S. Augmented anticancer efficacy of doxorubicin loaded polymeric nanoparticles after oral administration in breast cancer induced animal model Molecular Pharmaceutics 2011, 8, (4), 1140-51. 39. Jain, S.; Thakare, V. S.; Das, M.; Godugu, C.; Jain, A. K.; Mathur, R.; Chuttani, K.; Mishra, A. K. Toxicity of Multiwalled Carbon Nanotubes with End Defects Critically Depends on Their Functionalization Density. Chemical Research in Toxicology 2011, 24(11), 2028-2039 40. Bachilo, S. M.; Strano, M. S.; Kittrell, C.; Hauge, R. H.; Smalley, R. E.; Weisman, R. B. Structure-assigned optical spectra of single-walled carbon nanotubes. Science 2002, 298, (5602), 23612366. 41. Jia, G.; Wang, H.; Yan, L.; Wang, X.; Pei, R.; Yan, T.; Zhao, Y.; Guo, X. Cytotoxicity of carbon nanomaterials: single-wall nanotube, multi-wall nanotube, and fullerene. Environmental science & technology 2005, 39, (5), 1378-1383.

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42. Schneider, R.; Schmitt, F.; Frochot, C.; Fort, Y.; Lourette, N.; Guillemin, F.; Müller, J.-F.; Barberi-Heyob, M. Design, synthesis, and biological evaluation of folic acid targeted tetraphenylporphyrin as novel photosensitizers for selective photodynamic therapy. Bioorganic & medicinal chemistry 2005, 13, (8), 2799-2808. 43. Thomas, T. P.; Majoros, I. J.; Kotlyar, A.; Kukowska-Latallo, J. F.; Bielinska, A.; Myc, A.; Baker Jr, J. R. Targeting and inhibition of cell growth by an engineered dendritic nanodevice. Journal of medicinal chemistry 2005, 48, (11), 3729-3735. 44. Shanbhag, V. R.; Crider, A. M.; Gokhale, R.; Harpalani, A.; Dick, R. M. Ester and amide prodrugs of ibuprofen and naproxen: Synthesis, anti‐inflammatory activity, and gastrointestinal toxicity. Journal of pharmaceutical sciences 1992, 81, (2), 149-154. 45. Levine, R.M.; Clark, B.B. Relationship between struction and in vitro metabolism of various esters and amides in human serum Journal of Pharmacology and Experimental Therapeutics 1955, 113, (3), 272-282. 46. Aldridge, W. Serum esterases. 1. Two types of esterase (A and B) hydrolysing p-nitrophenyl acetate, propionate and butyrate, and a method for their determination. Biochemical Journal 1953, 53, (1), 110. 47. Aldridge, W. Serum esterases. 2. An enzyme hydrolysing diethyl p-nitrophenyl phosphate (E 600) and its identity with the A-esterase of mammalian sera. Biochemical Journal 1953, 53, (1), 117. 48. Landowski, C. P.; Vig, B. S.; Song, X.; Amidon, G. L. Targeted delivery to PEPT1overexpressing cells: acidic, basic, and secondary floxuridine amino acid ester prodrugs. Molecular cancer therapeutics 2005, 4, (4), 659-667. 49. Cho, K.; Wang, X.; Nie, S.; Shin, D. M. Therapeutic nanoparticles for drug delivery in cancer. Clinical cancer research 2008, 14, (5), 1310-1316. 50. Qu, G.; Bai, Y.; Zhang, Y.; Jia, Q.; Zhang, W.; Yan, B. The effect of multiwalled carbon nanotube agglomeration on their accumulation in and damage to organs in mice. Carbon 2009, 47, (8), 2060-2069. 51. King, P. D.; Perry, M. C. Hepatotoxicity of chemotherapy. The oncologist 2001, 6, (2), 162. 52. Fried, M.; Kalra, J.; Ilardi, C. F.; Sawitsky, A. Hepatocellular carcinoma in a long term survivor of acute lymphocytic leukemia. Cancer 1987, 60, (10), 2548-2552. 53. Singh, R. P.; Das, M.; Thakre, V. S.; Jain, S. Functionalization density dependent toxicity of oxidized multiwalled carbon nanotubes in a murine macrophage cell line. Chemical Research in Toxicology DOI: 10.1021/tx300228d 2012.

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Figures and Tables

Pristine-MWCNTs

HN

NH2 N

COOH NH

N

N

N O O

FA-NHS ester

OH

H2N

HN

AF

HN O

[50:1]

HN

O HN O

NH O NH O NH O NH2 O NH NH

O

FA

FA

O

O

HN O

O

H2N

H2N

NH2 excess, 12h

O

O

N

HN

O O EDBE

H 2N

H2N

H2N

(i) H2SO4/HNO3 [O] (ii) SOCl2, reflux, 24 h

HN

2

HN

NH NH2

2

Amine-MWCNTs

O HN O

AF-488/647)

OH O Glycidol

AF

rt, stirring, 18 h, dark O HO FA

O

FA

HO OH

HO

O NH

N

HN O

O HN

HN

HN

AF

O

H N O

O

FA

O MTX

O

MTX O O

N O HN

AF

48 h, stirring, dark

OH

HO OH

HN

Activated MTX

OH

O

O O

FA

EDC, DMSO Pyridine

NH2

OH

O

OH

OH HO

OH

N

NH2 N

OH

NH O NH O NH O NH N N FA O

OH N H

N N

COOH

O

N AF

HN

ETOH, Et3H rt, stirring, 12 h, dark

NH O NH NH O NH O NH NH2 NH NH 2 FA O O FA AF-FA-MWCNTs

HN

NH O

HN

FA

FA

NH

Technitium-99m

HN O

O

AF-647

O

AF O

H N

H N

O O

AF

O XTM

O O NH O NH NH NH N N FA O OH

O

O

O

MTX

OH

O N

OH

Tc (99m)

OH

OH

O O AF-FA-MTX-MWCNTs MTX O XTM O (1)

MTX

NH

O

HO

O

MTX

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. 26


Page 27 of 44

O

MTX

O O O HO O FA OH FA m-PEG O N NH HN HN O O HN HN O AF

O

N NH H O FA NH H2N

O

FA

OHHO

O MTX

O

NH OH

N

XTM MTX O

O

(1)

O H N

H N

FA O

O (2)

O

O HO

O

NH2

HN

FA MTX

O

O

HN O

OH O

XTM

AF MTX

O

O NH NH

O

NH O O NH O NH N FA O

O

HN

O

AF H N

H N

AF

NH

O

NH

MTX

O

MTX

O

O O

m-PEG

O O HO O OH

OH

MTX

m-PEG

O O

HO

N

AF

H2N

NH2 HN HN O O

O

O

AF H2N

H N

NH O O NH O NH2 N

O

NH N

O (4)

O

O O O

MTX

O

MTX

O

OCH3

n

MTX

OHHO

O

MTX

OH OH

O

O

O O

O

O

O

n

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(4)

(3)

OCH3

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

27


160

SLF A549 cell lysate Rat Plasma PBS

140 120

(a)

100 80 60 40 20 0

0

10

20

30

40

50

Cumulative MTX Release (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Cumulative MTX Release (%)


Page 28 of 44

160

SLF A549 cell lysate Rat Plasma PBS

140 120

(b)

100 80 60 40 20 0

0

10

20

Time (h)

30

40

Time (h)

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)

(b)

(i)

(ii)

50 µm

50 µm

50 µm

(ii)

(iii)

(i)

50 µm

(iii)

50 µm

50 µm

(A)

28


50

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(a)

(b)

(i)

(ii)

50 µm

50 µm

(i)

(iii)

50 µm

(ii)

50 µm

(iii)

50 µm

50 µm (B)

Figure 4. In vitro cell uptake studies: FR (+ve) A549 (A) and MCF 7 (B) cells were incubated with AF647-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.

(a)

(b)

(c)

(d)

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.

29



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Page 30 of 44

(A)

(i)

(ii)

r=0.64

(iii)

(iv)

(B)

(ii)

(i)

(iii)

r=0.28

(iv)

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.

30


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400

Tumor Volume (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Control FA-CNTs MTX 2 3 1

300

200

100

0 0

2

4

6

8

10

12

14

16

Time (Day) 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.

31



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Tables Table 1: Particle characteristics of functionalized MWCNTs Conjugate studied Oxidized MWCNTs EDBE-MWCNTs FA-MWCNTs FA-MTX-MWXNTs

Hydrodynamic size nm 173.8±4.2 230.9±3.7 361.3±8.7 413.1±10.3

Zeta Potential mV 0.421±0.052 0.437±0.034 0.280±0.021 0.411±0.036

PDI

-57.5±2.7 +9.2±0.6 -21.6±4 -24.5± 2.3

Particle size (Length) 1,2 nm 400-700 400-700 400-700 400-700

1

Diameter 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 Free MTX 3 1

IC-50 (µg/ml), A549 cells 7.26 5.32 2.13

IC-50 (µg/ml), MCF 7 cells 7.36 5.19 1.95

Table 3 (a) In vivo bio-distribution profile of free 99mTc-MTX Organ or tissue Blood Heart Lungs Liver Spleen Kidney Stomach Intestines Tumor Muscle Tumor: Muscle

0.5h 13.29 ±0.89 0.10±0.02 3.65±0.12 10.61±1.36 8.36±0.62 21.25±1.37 0.06±0.01 0.07±0.01 0.21±0.05 0.17±0.03 1.23

% ID /g recovered after 2h 4h 7.27±0.98 4.12±0.49 0.10±0.02 0.06±0.01 5.26±0.34 2.10±0.13 15.81±1.12 10.35±0.87 11.25±0.75 8.83±.65 25.15±2.65 29.64±2.47 0.08±0.02 0.07±0.01 0.10±0.02 0.08±0.01 0.23±0.02 0.26±0.05 0.14±0.06 0.09±0.02 1.64 2.88

32


24h 0.27±0.9 0.04±0.01 0.16±0.02 0.48±0.04 0.51±0.03 1.30±0.17 0.05±0.01 0.07±0.01 0.06±0.02 0.04±0.01 1.5

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Table 3 (b) In vivo bio-distribution profile of free 99mTc-AF-MTX-MWCNT (3) Organ or tissue Blood Heart Lungs Liver Spleen Kidney Stomach Intestines Tumor Muscle Tumor: Muscle

0.5h 0.74±0.03 0.23+0.02 24.65 ± 0.55 28.46 ±1.38 12.6 ± 0.2 1.87+0.13 0.53 ±0.03 0.66 ±0.05 1.68 ±0.12 0.44 ±0.08 3.81

% ID /g recovered after 2h 4h 0.62±0.03 0.46±0.02 0.17+0.04 0.15+0.06 19.2 ±0.4 15.8 ±0.7 16.2 ±0.7 14.26 ±0.03 6.71 ±0.03 5.65 ±0.45 0.92 ±0.04 0.63 ±0.05 0.49 ±0.01 0.42 ±0.02 0.54 ±0.02 0.32 ±0.02 1.92 ±0.18 2.02 ±0.12 0.36±0.07 0.17 ±0.01 5.3 5.94

24h 0.12±0.02 0.06±0.01 5.3 ±0.4 8.35±0.65 4.35 ±0.15 0.49 ±0.05 0.26 ±0.02 0.11 ±0.01 1.14 ±0.13 0.34 ±0.08 3.33

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

Organ or tissue Blood Heart Lungs Liver Spleen Kidney Stomach Intestines Tumor Muscle Tumor: Muscle

% ID /g recovered after 2h 4h 0.62±0.03 0.49±0.02 0.18+0.01 0.13+0.02 14.2 ±0.4 12.8 ±0.7 12.5 ±1.7 10.21 ±1.12 5.91 ±0.03 4.45 ±0.48 0.92 ±0.12 0.65 ±0.05 0.45 ±0.11 0.43 ±0.08 0.42 ±0.02 0.31 ±0.02 2.08±0.005 2.21 ±0.12 0.24 ±0.02 0.34 ±0.01 8.66 7.11

0.5h 0.83±0.03 0.23+0.02 22.65 ± 0.55 26.13 ±1.48 11.8 ± 0.2 1.69+0.18 0.52 ±0.03 0.65 ±0.05 1.92 ±0.16 0.38 ±0.08 4.52

24h 0.21±0.02 0.05±0.01 4.3 ±0.4 7.16 ±0.69 3.15 ±0.15 0.47 ±0.05 0.26 ±0.02 0.14 ±0.01 1.16 ±0.03 0.30 ±0.02 3.86

Table 3 (d) In vivo bio-distribution profile of free 99mTc-MTX-FA-AF-MWCNTs (1) Organ or tissue Blood Heart Lungs Liver Spleen Kidney Stomach Intestines Tumor Muscle Tumor: Muscle

0.5h 0.61±0.03 0.19±0.03 23.3 ± 0.4 26.95±0.35

12.13 ± 0.25 1.54±0.07 0.46±0.06 0.23±0.02 3.28±0.02 0.60±0.09 5.46

% ID /g recovered after 2h 4h 0.46±0.03 0.34±0.05 0.12±0.01 0.09±0.004 1.57±0.11 1.275±0.045 9.165±0.465 7.831±0.241 3.66±0.37 2.84±0.075 0.63±0.06 0.465±0.015 0.36±0.03 0.29±0.07 0.17±0.02 0.133±0.05 4.44±0.005 4.46±0.121 0.252±0.005 0.22±0.02 17.61 22.09 33


24h 0.285±0.025 0.03±0.02 0.945±0.035

5.3 ±0.4 0.78±0.03 0.12±0.01 0.055±0.005 0.04±0.01 2.56±0.26 0.096±0.005 26.66


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table of Contents Use Only Augmented Anticancer Activity of a Targeted, Intracellularly Activatable, Theranostic Nanomedicine based on Fluorescent and Radiolabeled, Methotrexate-Folic acidMultiwalled Carbon Nanotube Conjugate Manasmita Das, Satyajit R. Datir, Raman Preet Singh, Sanyog Jain*

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476x688mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

476x549mm (96 x 96 DPI)


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


476x198mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

476x320mm (96 x 96 DPI)


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


476x323mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1375x325mm (96 x 96 DPI)


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


317x300mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

370x342mm (96 x 96 DPI)


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


317x267mm (96 x 96 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

555x406mm (96 x 96 DPI)


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Augmented Anticancer Activity of a Targeted ... - ACS Publications

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