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Conjugation of docetaxel with multiwalled carbon nanotubes and co-delivery with piperine: Implications on pharmacokinetic profile and anti-cancer activity. Kaisar Raza, Dinesh Kumar, Chanchal Kiran , Manish Kumar, Santosh Kumar Guru, Pramod Kumar, Shweta Arora, Gajanand Sharma, SHASHI BHUSHAN, and O. P. Katare Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00183 • Publication Date (Web): 16 May 2016 Downloaded from http://pubs.acs.org on May 19, 2016

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Molecular Pharmaceutics

Conjugation of docetaxel with multiwalled carbon nanotubes and co-delivery with piperine: Implications on pharmacokinetic profile and anti-cancer activity Kaisar Raza,1* Dinesh Kumar,1 Chanchal Kiran,1 Manish Kumar,1 Santosh Kumar Guru,2 Pramod Kumar,1 Shweta Arora,3 Gajanand Sharma,4 Shashi Bhushan,2 and O. P. Katare4 1Department

of Pharmacy, School of Chemical Sciences and Pharmacy, Central

University of Rajasthan, Bandar Sindri, Distt. Ajmer, Rajasthan, India-305817 2Division

of Cancer Pharmacology, Indian Institute of Integrative Medicine, Jammu,

India-180001 3Department

of Biotechnology, Banasthali Vidhyapith University, P.O. Banasthali

Vidhyapith, Rajasthan-304022 4Division

of Pharmaceutics, University Institute of Pharmaceutical Sciences, Panjab

University, Chandigarh, India-160014

*Corresponding Author Dr. Kaisar Raza Department of Pharmacy School of Chemical Sciences and Pharmacy Central University of Rajasthan Bandar Sindri, Distt. Ajmer, Rajasthan, India-305 817 E-mail: [email protected]; [email protected]

ACS Paragon Plus Environment


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Abstract Nanotechnology-based drug products are emerging as promising agents to enhance the safety and efficacy of established chemotherapeutic molecules. Carbon nanotubes (CNTs), especially multi-walled CNTs (MWCNTs) have been explored for this potential, owing to their safety and other desired attributes. Docetaxel (DTX) is an indispensable anticancer agent, having wide applicability in variety of cancers. However, the potential of DTX is still not completely harvested due to problems like poor aqueous solubility, low tissue permeability, poor bioavailability, high first pass metabolism and dose-related toxicity. Hence, it was proposed to attach DTX to MWCNTs and co-administer it along with piperine with an aim to enhance the tissue permeation, anti-cancer activity and bioavailability. The FT-IR, UV and NMR spectroscopic data confirmed successful conjugation of DTX to MWCNTs and adsorption of piperine onto MWCNTs. The co-delivery MWCNT-based system offered drug release moderation and better cancer cell toxicity than that of plain DTX as well as DTX–CNT conjugate. The pharmacokinetic profile of DTX was exceptionally improved by the conjugation, in general and co-administration with piperine, in specific vis-à-vis plain drug. Hence, the dual approach of MWCNTs conjugation and piperine co-administration can serve as a beneficial option for enhancement of the performance of DTX in cancer chemotherapy. Keyword: Nanomedicine, Drug delivery, Bioavailability, Adsorption, Drug release, CYP3A4 inhibition, MCF-7, MDA-MB-231, IV Push


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1.

Introduction Nanotechnology inherits the potential to develop smart drugs to selectively target the cancer cells and also offer promises to load combination of drugs onto a single nano-sized carrier for both therapeutic and diagnostic purpose.1 Nanotechnologybased carriers are now being explored for the better and safer drug delivery in medical sciences.2 The potential of cancer nanotechnology depends on the quality of engineered vehicles including nano size, which controls the tumor penetration and specificity.3 Examples of such carriers are vesicular systems, micelle-based colloidal vehicles, particulate nanocolloids, emulsified nanoconstructs, fullerenes and carbon nanotube (CNTs).4,5,6 The drugs have either been conjugated or incorporated with in these nanocarriers.7 Size of these nanomaterials is 100 to 1,000 folds smaller than mammalian cells including cancer cells, hence, they can easily deliver drugs by oral and i.v. routes to the desired sites.8

Therefore, the drug delivery potential of

nanomaterials, especially to the cancer cells, is regarded as a promising tool to enhance the delivery options.7 Nanotechnology has proved its worth to improve the cancer chemotherapy by destroying cancer cells with minimal damage to normal cells, as well as by detection and elimination of cancerous cells prior to tumor formation.10 Some nanotechnology-based anticancer formulations have been marketed, while a large number of similar products are in the preclinical and clinical studies, as shown in Table 1. Space for Table 1 CNTs is a class of carbon nanostructures along with fullerenes, generally regarded as the third crystalline allotropic form of sp2-hybridized carbon atoms.20 Since, the exploration of CNTs in nanotechnology, their inherent physicochemical properties such as high aspect ratio and surface area, easiness of drug loading via ̟−̟ stacking, high mechanical strength, and photo acoustic effect have made them potential contender for targeted drug delivery and imaging.11,21 CNTs also possess high adsorption properties, high drug conjugation potential, high thermal and electrical conductivities and substantial chemical stability.22 On the other hand, most of the



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anticancer chemotherapeutic agents inherit several challenges like dose-dependent side effects, low aqueous solubility, less tissue specificity, poor stability, shorter halflives and compromised tissue penetration.21 In this regards, CNTs are being explored to enhance the outcomes of cancer chemotherapy. These carriers are reported to possess the potential to deliver the drugs in a targeted and sustained manner to the cancerous sites.12,24 CNTs can modify the drug delivery properties by physical adsorption of drug within or on the surface the CNTs, or by noncovalent/covalent interaction of drug molecules, peptides, nucleic acids to the inserted functional groups of the modified CNTs or by the passive diffusion and endocytosis of drug-loaded CNTs into the target site.25 Docetaxel (DTX) is class of anticancer agents, which belongs to the taxane family. DTX was discovered in the 1980 and is reported to possess higher potency than that of paclitaxel, as an anticancer agent. DTX is used in the chemotherapeutic management of various malignant cancers such as breast cancer, ovarian carcinoma, lung cancer, and head or neck cancer.26 Mechanism of action of DTX involves its binding to the β-subunit of tubulin protein of the microtubules resulting the hyperstabilization of microtubule assembly, which results in the desired anticancer activity.27 The drug has also been approved by U.S. FDA as an injectable product, Taxotare@ for use in combination with cisplatin and fluorouracil for cancer chemotherapy. It is an important second generation semisynthetic anticancer drug, belonging to biopharmaceutical classification system (BCS) class IV with limited solubility and permeability.28 The major challenges with DTX are hepatic metabolism, poor aqueous solubility and poor bioavailability (95% purity, L-0.5–2 µm, Φ-20–50 nm) were supplied by M/s Nanostructured and Amorphous Materials Inc, Houston, USA. Dialysis membrane was purchased from M/s Himedia Laboratories, Pvt, Ltd, Mumbai, India. Buffer ingredients were purchased from M/s CDH Co. Ltd, New Delhi, India. Oxidizing acids and solvents were purchased from M/s Molychem Co. Ltd., Mumbai, India, and M/s Thermo Fisher Scientific India Co. Ltd., Mumbai, India, respectively. HPLC column, Oyster ODS3 (250-4.6 mm, 5µm) was purchased from M/s Merck Specialities Private Limited, Mumbai, India. The MCF-7 and MDA-MB-231 cell lines were procured from European Collection of Cell Cultures (ECACC), a Culture Collection of Public Health, England. Distilled water was used during the studies and all the chemicals/reagents were employed as such, without any further purification.

2.2

Methods

2.2.1 Synthesis of MWCNTs-DTX conjugate



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The synthetic scheme for conjugation of DTX with MWCNTs has been depicted as Figure 1. Pristine MWCNTs, 200 mg were added to the mixture of conc. H2SO4 and conc. HNO3 (3:1 volume ratio; 50 mL). The resulting reaction mixture was sonicated for 10 min with 30 seconds interval, using the ultrasonic probe sonicator, followed by stirring for 24 hours at room temperature. Upon completion of reaction, 800 mL distilled water was added to the reaction mixture and the diluted dispersion was kept overnight for sedimentation. Water was decanted and remaining dispersion was vacuum filtered using Whatman filter paper (0.22 µm). The washings with deionized water were performed repeatedly till the filtrate pH reached neutral value. The residue was dried at 60ºC for 2 h to fetch with carboxylated MWCNTs (fMWCNTs). f-MWCNTs (150 mg) in SOCl2 (15 mL) and tetrahydrofuran (THF; 8 mL) were refluxed at 80 ºC for 36 h and vacuum filtered, after completion of reaction. Thereafter, residue was washed with THF and dried under vacuum desiccator at room temperature for 30 minutes to give acylated MWCNTs.32 Acylated MWCNTs, (20 mg) were dispersed in mixture of 1.5 mL anhydrous tetrahydrofuran (THF) and 3 mL pyridine, by water bath sonication for 20 minutes. DTX (20 mg) was added to the dispersion and the reaction mixture was stirred for 24 hours, under nitrogen environment at room temperature. The reaction progress was monitored using thin layer chromatography. Reaction mixture was vacuum filtered, washed with THF to remove the unconjugated drug and was air dried.32 Space for Figure 1 2.2.2 Determination of extent of carboxylation The number of carboxylic acid groups (-COOH) on f-MWCNTs were determined by acid-base titration. f -MWCNTs (10 mg) were dispersed in 20 mL of distilled water by sonication. Dispersion was titrated against 0.005 M standardized NaOH solution, using phenolphthalein as indicator (n=3).12 2.2.3

Determination of extent of drug loading


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The concentration of unconjugated DTX was determined by high performance liquid chromatography (LC-2010C HT, M/s Schimadzu Co. Ltd., Chiyoda-ku, Tokyo, Japan) with the conditions as: Merck HPLC Column, Oyster ODS3 (250-4.6 mm, 5µm); Mobile phase acetonitrile: water 50: 50 v/v; Column temperature 30ºC; Detection wavelength 231 nm; Flow rate 1.0 mL/min; Injection volume 20 µL; Run time 30 minutes and Detector used PDA (SPD-M20A). Repeated washings of THF of the last step of conjugation were collected and solvent was evaporated on rotatory-evaporator (R-215, M/s Buchi, Flawil, Switzerland). To the residue, required amount of ACN was added. Test samples of DTX were run in the HPLC system, as per the earlier reported protocol.5 2.2.4 Physical adsorption of piperine on carboxylated f-MWCNT The extent of physical adsorption of piperine at different concentrations, i.e., 0.1 mg/mL, 0.2 mg/mL, 0.4 mg/mL, 0.6 mg/mL, 0.8 mg/mL, and 1 mg/mL was observed on 0.2 mg/mL of f-MWCNTs. f-MWCNTs and piperine were added in ethanol and mixture was sonicated for 20 minutes, using water bath sonicator. Resulting dispersions were kept overnight and filtered using 0.22 µm Whatman filter paper. The optical density of the clear supernatant was recorded at λmax of the drug to determine the extent of absorption. 2.2.5

Characterization and evaluation

2.2.5.1

FT-IR Fourier Transform Infrared Spectroscopy (FT-IR) was used for the analysis of functional groups on the compounds. The samples and potassium bromide were mixed thoroughly in the mass ratio of 2:98 and punched to a tablet, employing hydraulic press. The FT-IR data were recorded using FTIR Spectrometer (M/s Perkin Elmer Co., Waltham, Massachusetts, USA) at a wave number range of 4000 cm-1 to 400 cm-1.

2.2.5.2

NMR Analysis



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Saturated solutions in deuterated chloroform (CDCl3) were used to record the

13C

NMR on Avance II 400 NMR Spectrometer (M/s Bruker Bio Spin

Corporation, Indiana, USA). Drop of CS2 was employed to dissolve the DTX-MWCNTs-conjugate. 2.2.5.3

Scanning Electron Microscopy (SEM) JEOL Scanning Electron Microscope (JSM 6490 LV, M/s Hitachi, Tokyo, Japan), installed at University Science Instrumentation Centre (USIC) in Baba Sahib Bhimrao Ambedkar University, Lucknow, Uttar Pradesh, India was employed for the study. In brief, after cleaning the samples by compressed gas, the samples were dried. Dispersion of powder in polar solvent was placed on glass slide and evaporated. Photographs were recorded at suitable magnification.

2.2.5.4

Particle size distribution and zeta potential studies Particle size and poly dispersity index (PDI) of the developed systems were determined using Malvern Zetasizer (M/s Malvern, Worcestershire, UK) installed at Dr. S. S. Bhatnagar University Institute of Chemical Engineering and Technology, Panjab University, Chandigarh. Zetapotential of the nanoconstructs dispersed in phosphate buffer saline, pH 7.4 (10 mg/10mL) was also determined using the same equipment. The carriers were dispersed in water for injection. The sample was sonicated for 15 min on water bath sonicator for proper dispersion of the studied samples. The average of three repeated value for each sample was reported as the ﬁnal result for the test samples.

2.2.5.5

In-vitro drug release studies In vitro drug release studies of the samples (DTX, piperine, DTX-MWCNT and piperine-DTX-MWCNT) were carried out in 0.5% sodium dodecyl sulphate solution in phosphate buffer saline pH 5.8 (PBS). In dialysis bag, 1 mL of each sample dispersed in the diffusion medium was placed with


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DTX/piperine equivalent concentration of 10 mg/mL. The temperature was maintained at 37±1° C along with stirring. Aliquots of 0.5 mL were withdrawn at different time intervals and equal volume of fresh diffusion medium was added to maintain the sink conditions.25 The samples were analyzed by RP-HPLC. In-vitro drug release data was fitted to various release models, such as zero-order, first-order, Higuchi equation and Korsemeyer–Peppas model. Regression analysis was formed to find the best fitted order and the release kinetics.12 2.2.5.6

Ethical compliance All the animal protocols were duly approached by Institutional Animal Ethics Committee, Panjab University, Chandigarh, India (Ref. no. IAEC/411) and the studied were performed in strict adherence to the ethical guidelines.

2.2.5.7

Ex-vivo hemolysis studies Ex-vivo hemolysis studies were performed on blood samples from healthy Wistar rats (200-250g; 4-6 weeks old). Retro-orbital plexus of the rats was used to collect the blood samples. Blood (0.5 mL) was transferred in a vial containing sodium citrate solution. The blood was centrifuged to fetch with pellet of erythrocytes. Saline solution was used to wash the RBC pellet. The harvested RBCs were re-suspended in saline and 2% (w/v) formulation (docetaxel, pristine MWCNTs, DTX-MWCNT conjugate, piperine and DTX-MWCNTs conjugate along with piperine) was added to it. One test tube containing RBCs suspended in double distilled water served as a control. All the test tubes were incubated for 1 hour at 37°C with gentle shaking. After completion of incubation, centrifugation was done for 5 minutes at 2000×g. Clear supernatant was analyzed spectrophotometrically at 415 nm. In order to determine the % hemolysis, the hemolysis induced with double distilled water was taken as 100%.5



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2.2.5.8

In-vitro anti-cancer activity In-vitro anti-cancer studies were performed on MCF-7 and MDA-MB-231 human breast cancer cells. Briefly, 6 × 103 cells were seeded in 96 well plates and were treated with various concentrations of the test material (equivalent to 1 µg/mL and 10 µg/mL of DTX) and incubated for 48 hours. MTT (2.5 mg/mL), 20 µL was added for 4 hours before the termination of the experiment. The plates were centrifuged at 400 × g for 15 minutes and formulated MTT formazan crystals were dissolved in 150 µl of DMSO. The absorbance of the solution was measured at 570 nm with reference wavelength as 620 nm.5 The IC50 values were determined, using GraphPad Prism Version 5.0 software.

2.2.5.9

In-vivo pharmacokinetic studies Pharmacokinetic studies were performed on Unisex Wistar rats (200-250 g; 4-6 weeks old). Rats were divided into three groups, based on the number of samples, with three animals in each group. Group 1 animals received plain DTX, Group 2 animals received MWCNT-DTX conjugate, while the animals of Group 3 received mixture containing MWCNT-DTX conjugate and physically adsorbed piperine on f-MWCNTs. Respective treatments were injected to the animals of all three groups after dispersing in “water for injection”, aseptically. Dose equivalent to 5 mg/kg (0.3 mL) was aseptically injected into the tail vein of the rats. Blood samples (0.3 mL each time) at specified time intervals were collected. Retro-orbital plexus mode was employed for blood sampling. To the samples collected, methyl tert-butyl ether (2 mL) was added and centrifuged (2000 ×g). Supernatant was collected, air dried at 40°C and re-dissolved in ACN. Syringe filter (0.22 µm) was employed to filter the samples for the analysis of drug by RP-HPLC.12 Different pharmacokinetic parameter like (AUC)0-12, (AUC)0∞,

K, Vd, t

1/2

and Cl were determined using 1CBM iv push model, using

MS office Excel.


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3

Results and discussion

3.1

Extent of carboxylation The results revealed the presence of ≈ 2.41 x 1018 (0.0004 milli equivalent) of COOH per mg of the f-MWCNTs. This crucial data inferred that approx. same milli equivalents of DTX were required for efficient conjugation of –OH of DTX to the -COOH groups of f-MWCNTs. Hence, it was decided to carry over the conjugation step with 1:1 mass ratio of f-MWCNTs to DTX for efficient conjugation.32

3.2

FT-IR analysis Figure 2A represents the FT-IR spectrum of pristine MWCNTs. FT-IR data as represented in Figure 2B, shows –OH stretching of carboxylic group (-COOH) at 3463.3 cm-1 and the stretching of C=O at 1702.66 cm-1, indicating successful carboxylation. In Figure 2C, stretching of C-Cl at 666.76 cm-1 with disappearance of –OH stretching of carboxylic group indicates successful acylation of carboxylated MWCNTs. The FT-IR spectrum of pure DTX has been shown as Figure 2D. Disappearance of C-Cl stretching and stretching of C=O was observed at 1620.92 cm-1 in the drug-CNT conjugate (2E). The FT-IR spectrum of each product and the comparison between the various products inferred successful formation of the desired compounds. The findings are in consonance with previous published reports on CNT-conjugates.32 Space for Figure 2

3.3

NMR analysis Results of

13C

NMR spectra have been shown as Figure 3. In

13C

NMR, the

peak appearing at δ 128.58, 130.73 corresponded to (C=C) and in the range of 0 to 38.83 ppm represented the saturated carbons of pristine MWCNTs. In case of DTX-MWCNT conjugate, the peak at δ of 192.610 reflected (C=O), 129.78 to 94.16 indicated presence of aromatic (C=C), 59.58 confirming (-OCH2-) and 38.17 to 1.104 inferred saturated carbons. Henceforth, the analysis of 13C NMR



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spectra confirmed the successful synthesis of the desired DTX-MWCNT conjugate. Space for Figure 3 3.4

Extent of drug loading The extent of DTX loading on acylated MWCNTs was found to be 62.8± 4% w/w. The studies confirmed the substantial drug loading potential of MWCNTs. However, the desired complete conjugation of DTX onto the acyl groups of MWCNTs could not been achieved, plausibly due to the closely located –COOH groups and the steric hindrance between two DTX molecules. The observed drug loading in the present studies is greater than the previously reported loading of 54%.32 The plausible reason might be the quantification of free carboxyl groups on the f-MWCNTs and subsequent attachment of DTX in the desired stoichiometry.

3.5

Physical adsorption of piperine on f-MWCNTs Results of physical adsorption of piperine on f-MWCNTs showed an increase in the percent drug adsorption with increase in f-MWCNTs to drug ratio. However, after 1:3 mass ratio f-MWCNTs to piperine (~54% adsorption of drug), there was no significant increase in adsorption, indicating saturation.34 Such kind of behavior is characteristic for physical adsorption, which is limited after occupancy of the active sites of the adsorbent, as per the Freundlich’s-adsorption isotherm. Henceforth, mass ratio of 1:3 for fMWCNTs to piperine was selected for further studies.

3.6

Scanning Electron Microscopy (SEM) Figure 4 (A-D), show the SEM images of pristine MWCNTs (4A), carboxylated MWCNTs (4B), DTX-MWCNTs conjugate (4C) and physically adsorbed piperine onto f-MWCNTs (4D). Carboxylated carbon nanotubes appeared to be less clustorous and smaller in size vis-à-vis pristine MWCNTs. This indicated the distortion of nanotubes lengths, after oxidative treatment,


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as already reported by various researchers.32 The findings are in agreement with the previously published literature on similar drug carriers.42 Space for Figure 4 3.7

Particle size distribution and zeta potential The micromeretics and zeta potential data have been shown in Table 2. A conspicuous decrease in particle size, after the carboxylation, from 461.5 nm to 282.4 nm was observed. After oxidative carboxylation with strong acids, decrease in average size of MWCNTs is an established fact due to breaking of the basic pristine structure. 32 The results obtained are in close agreement with the results from SEM studies. Increase in particle size was observed after drug conjugation, which virtually indicated the potential drug conjugation on the MWCNTs.33 The PDI values were found to be range in between 0.139 to 0.354. These findings might be ascribed to the non-symmetric nature of CNTs.34 The obtained PDI values are less than 0.4, advocating appreciable reliability as well as a bit of homogeneity in the reported size values. Space for Table 2 The surface charge on pristine MWCNTs and on f-MWCNT was found to be negative. The increase in the surface negative charge of f-MWCNTs can be attributed to the presence of acidic group (-COOH).11 The observed zeta potential values of carboxylated MWCNTs were appreciably good, of the order of -39.5 mV, advocating substantial dispersion stability. The zeta potential of the final conjugate was around -19.3 mV and satisfactory enough, as the conjugate was to be stored in solid form, which needed to be dispersed aseptically in “water for injection”, just prior to use.

3.8

In vitro drug release studies Figure 5 shows the drug release profile of DTX and piperine from the studied systems. An initial burst release of drug was observed from the developed



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systems (~ 13% in initial 30 minutes). However, for longer duration, the potential of controlled drug release of the conjugate is clearly visible from the in vitro release data. In a span of 24 hours, only 63.9% of the loaded drug was released from the conjugate, while the release from pure drug in this period was about 87.5%. On the other hand, the conjugate released around 70.0% of adsorbed piperine against 92.7% from the plain drug. The significant difference in the drug release pattern from the conjugate vis-à-vis the plain drug(s) indicated potential application in drug delivery, as the MWCNTs were able to control the drug release pattern. The conjugation of DTX with MWCNTs was chemical in nature and the bond was of ester linkage. The esterase enzymes, as well as the acidic pH, of cancer cells will cleave the ester bond, at the desired site and release the drug to exhibit its action. However, the f-MWCNTs system was also able to regulate the piperine release (approx. 94% adsorbed drug in 40 h) in the studied microenvironment. At every time point, the amount of piperine released was approximately 1.3-1.5 times higher than amount of DTX release till 12 h. This pattern was desired, as the prior release of piperine will execute its enzyme inhibitory action to facillate enhanced DTX concentration at the site of action.31 Equations of line and the values of coefficient of determination for release model fitting have been shown in Table 3. From the analysis of the data, it was inferred that the release pattern of both the drugs from the conjugate exhibited Higuchi release profile, while the drug release from the plain drug was observed to follow first-order release. As CNTs can be considered to be matrix for the drug, hence, Higuchi profile is expected from such carriers and is in consonance with the previous reports.12 Most of the advanced drug delivery devices and carriers have been reported to follow the Higuchian drug release profile and the same is believed to be the most practical release profile by the colloidal drug carriers.35 Space for Figure 5 and Table 3 3.9

Ex-vivo hemolysis studies


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To evaluate the safety profile of the developed nano-conjugates, the % hemolytic activity was determined and the findings have been shown as Figure 6. All the values of % hemolysis were found to be less than 5%. Therefore, the developed system can be regarded as blood compatible. The results are of great importance for the present conjugate, as it is intentioned to be res-suspended in “water for injection” and to be injected in the veins for the desired outcomes. Lower hemotoxicity ensured the biocompatibility of the developed system at the central compartment, i.e., blood. Space for Figure 6 3.10

In-vitro anti-cancer activity MTT anticancer assay on MCF-7 and MDA-MB-231 human breast cancer cells was performed and the results have been shown as Figure 7 and Figure 8. The % cell symmetry has been shown in Figure 9. The ratio of both the systems, i.e., DTX-MWCNTs and MWCNT-adsorbed piperine, employed was 1:1 w/w (0.24 mg of DTX and 0.81 mg piperine per mg of mixture). For comparison purpose, the concentrations of the DTX employed were 1 µg/mL and 10 µg/mL, and the corresponding piperine concentrations were 3.38 µg/mL and 33.75 µg/mL. At concentration of 1 µg/mL, the MWCNTs were able to enhance the drug efficacy of DTX by around 2.4 times, while at concentration of 10 µg/mL the anticancer activity was enhanced by approx. 2.7 times against MCF-7 cell lines. The results on the invasive cells, i.e., MDA-MB-231 cells, were also encouraging, as the conjugate offered around 4.2 times efficacy at concentration of 1 µg/mL, while around 1.9 times at the concentration of 10 µg/mL vis-à-vis drug. The incorporation of piperine further enhanced the cytotoxicity of DTX, as inferred from the decline in the IC50 values. The IC50 values for DTX, piperine, DTX-MWCNTs, and DTXMWCNTs-Piperine on MCF-7 cell lines were observed to be 25 µg/mL, 121 µg/mL, 14 µg/mL and 8 µg/mL, resp., whereas on MDA-MB-231 the values obtained were 15 µg/mL, 72.6 µg/mL, 10 µg/mL and 6 µg/mL, resp.



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However, the MWCNTs were observed to be non-cytotoxic in nature on the studied cells. The enhancement in the cytotoxic potential of the co-delivered system can be outcome of serious of processes like better adhesion of the conjugate due to CNTs, better penetration of the system by virtue of CNTs and piperine, release of adsorbed piperine, inhibition of CYP3A4 enzyme, hydrolysis of the ester linkage of MWCNT-DTX, release of DTX and ultimately the cell death.31 The enhanced cytotoxicity of DTX on both invasive and non-invasive cell lines by MWCNTs/piperine co-delivery corroborates the promises offered by this inorganic carrier. There were no substantial symmetrical changes in the cell symmetry after incubation during the test, as shown in Figure 9. This assured the reliability of the MTT assay, as the artifacts from the detached cells were not considered. Space for Figure 7, Figure 8 and Figure 9 3.11

In-Vivo pharmacokinetic evaluation The concentration versus time profile (Figure 10) showed a bit inclination towards 2-compartment open body model (2 CBM). But after plotting the log C versus time, the r2 values were ensured a good fit; hence, the data was forced for the execution, as per one compartment open body model (1 CBM). The derived equations of the semi-logarithmic plot have also been shown in the figure. The plasma concentrations of the drug in group receiving pure DTX dropped quickly, as clear from Figure 10. However, the decline in plasma DTX concentration from DTX-MWCNT conjugate administered group was relatively slower. Surprisingly, the group receiving DTX-MWCNT conjugate along with piperine showed the slowest decline in the plasma concentrations of DTX. The conjugate with MWCNTs offered approx. 2.6 times enhancement in AUC and co-administration of conjugate with piperine offered about 6.4 folds increment in the AUC of DTX vis-à-vis the pure drug, as shown in Table 3. The enhancement in AUC can be ascribed as the outcome of two processes,


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viz. delayed clearance by CNTs and CYP3A4 enzyme inhibition by piperine.31 The MWCNT conjugate was able to decrease the drug clearance to approx. 50%, while co-administered regimen decreased the same upto 20% in comparison to the drug clearance of the plain drug. Henceforth, the residence in the biological system was enhanced substantially by the conjugate and further improved by the piperine co-administered system. The findings are encouraging and offer a hope for the desired modulation of the pharmacokinetic profile of this bioavailability compromised, but promising anticancer agent. 4

Conclusions The conjugate of DTX to MWCNTs, in general and its co-administered regimen with piperine, in specific, were not only able to enhance the anti-cancer activity of DTX, but also offered substantial AUC enhancement and bio-residence to the drug. This approach offers a surfactant free system for DTX with a potential to alleviate the challenges associated with the drug. The in vitro and pre-clinical findings are encouraging and also establish the cell compatibility of MWCNTs. The same can be extrapolated and be utilized to develop a product based on dual approach of MWCNT conjugation and partial inhibition of CYP450 3A4.

5

Acknowledgements: The authors acknowledge the financial support from the University Grants Commission, New Delhi, India in the form of UGC-Start-Up Grant to the corresponding author. The authors also acknowledge Dr Ranjeet Kaur for her assistance in NMR operation and interpretation. This work would have been impossible without the gift sample of docetaxel from M/s Fresenius Kabi Oncology Limited, Gurgaon, India. Their support for the academic cause is highly appreciated.

6

Conflict of interest: The authors report no conflict of interest.

7

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Table 1: Clinical status of various nanocarriers in cancer chemotherapy S. No Nanocarrier Type 1. Inorganic nanomaterials

Clinical status Preclinical

2.

Polymeric micelles

Clinical trails

3.

Liposomes

Marketed

4. 5.

Albumin-Based particles Dendrimers

Marketed Clinical trial

Example Carbon nanotubes, Fullerenes, Silica particles, Gold particles11,12 Genexol-PM, SP1049C, NK911, NK01213,14 ,15 DaunoXome, Doxil®14, LIPUSU®17 Abraxane18 DEP™-Docetaxel19

Table 2: Tabular representation of particle size, PDI and zeta potential of MWCNTs, f-MWCNTs, acylated MWCNTs and DTX-MWCNTs conjugate, Piperine-MWCNTS and DTX-MWCNTs-Piperine conjugate (n=3) S. No.

Sample

Particle size (nm)

PDI

461.5 ±8.0 282.4 ± 6.1 451.4 ± 10 271.2 ±7.6

0.139 0.131 0.224 0.215

5.

MWCNTs f-MWCNTs Acylated MWCNTs DTX- MWCNT conjugate Piperine-MWCNTs

Zeta Potential (mV) -17.3 ± 4.8 -39.5 ± 8.6 6.41 ± 0.4 -7.10 ± 0.4

291.9 ± 8.7

0.263

-18.6 ± 2.2

6.

DTX-MWCNTs-Pip

287.9 ± 10.1

0.354

-19.3 ± 1.7

1. 2. 3. 4.



1 2 3 4 5 6 7 Table 3: Results of model fitting for the data obtained from release studies. 8 9 Parameter Zero Order First Order Higuchi Model 10 11 DTX Piperine DTX- CNTsDTX Piperine DTX- CNTsDTX Piperine DTX12 CNTs Piperine CNTs Piperine CNTs 13 Interceprt 162.08 36.67 9.35 32.93 1.08 1.80 2.38 1.80 19.30 21.07 7.80 14 Slope 51.35 2.49 x 6.94 2.41 x 0.13 -6.00 x -0.11 -4.00 x 1.19 1.69 0.82 15 10-2 10-2 10-4 10-4 16 2 0.8125 0.5489 0.8971 0.5706 0.9846 0.9734 0.8229 0.8248 0.8721 0.7437 0.9902 17R 18 19 20 Table 4: Tabular representation of different pharmacokinetic parameters 21 22 23 Parameter Unit DTX DTX-MWCNT 24 25 26 [‫]ܥܷܣ‬ଵଶ µg.mL-1.min 745.07 1882.85 ଴ 27 28 [‫]ܥܷܣ‬ஶ 907.90 2356.16 µg.mL-1.min 29 ଴ 30 31 K min-1 2.764 x 10-3 2.303 x 10-3 32 33 Vd L 0.337 0.167 34 35 Cl mL/min 1.006 0.461 36 37 t 1/2 min 250.07 301 38 39 40 41 42 43 44 45 46 ACS Paragon Plus Environment 47 48

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Korsmeyer Peppas Model CNTsPiperine 18.04 1.63 0.9833

DTX

Piperine

1.44 1.00 x 10-4 0.4688

1.30 3.00 x 10-4 0.3140

DTX-MWCNTPiperine 3046.85 5778.20 1.152 x 10-3 0.186 0.214 601.17

DTXCNTs 1.40 1.00 x 10-4 0.5987

CNTsPiperine 1.24 3.00 x 10-4 0.3226

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Figure 1: Synthetic scheme for the conjugation of DTX to MWCNTs.



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Figure 2: FT-IR spectrum of: (A) MWCNTs; (B) Carboxylated MWCNTs; (C) Acylated MWCNTs; (D) Plain DTX; and (E) DTXMWCNTs conjugate


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B

Figure 3: 13C-NMR spectrum of: (A) MWCNTs and (B) DTX-MWCNT conjugate



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Figure 4: SEM images of: (A) pristine MWCNTs; (B) f-MWCNTs; (C) MWCNT-DTX conjugate; and (D) physically adsorbed piperine on f-MWCNTs.


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Figure 5: Graphical representation of % drug release from various systems



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Figure 6: Bar diagram depicting the effect of various formulations on the RBCs


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Figure 7: Graphical representation of the cancer % cell death by various treatments on MCF-7 (A) and MDAMB-231 (B) cell lines.



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Figure 8: Microphotographs of (A) MDA-MB-231 cell lines, Control; (B) MWCNTs (10 µg/mL) treated MDA-MB-231 cell lines; (C) DTX (10 µg/mL) treated MDA-MB-231 cell lines; (D) Piperine (33.75 µg/mL) treated MDA-MB-231 cell lines; (E) MWCNT-DTX (10 µg/mL)MDA-MB-231 cell lines; (F) MWCNTs-DTX-Piperine (DTX = 10 µg/mL/ Piperine = 33.75 µg/mL) treated MDA-MB-231cell lines; (G) MCF-7 cell lines, Control; (H) MWCNTs (10 µg/mL) treated MCF-7 cell lines; (I) DTX (10 µg/mL) treated MCF-7 cell lines; (J) Piperine (33.75 µg/mL) treated MCF-7 cell lines; (K) MWCNTDTX (10 µg/mL) treated MCF-7 cell lines; (L) MWCNT-DTX-Piperine (DTX = 10 µg/mL/ Piperine = 33.75 µg/mL) treated MCF-7 cell lines.


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Figure 9: Bar diagram depicting the changes in the symmetry after the assigned treatment. Data expressed as mean ± SD of three similar experiments. (A) MDA-MB-231 cell lines, Control; (B) MWCNTs (10 µg/mL) treated MDA-MB-231 cell lines; (C) DTX (10 µg/mL) treated MDA-MB-231 cell lines; (D) MWCNT-DTX (10 µg/mL)MDA-MB-231 cell lines; (E) MWCNTs-DTX-Piperine (10 µg/mL ) treated MDA-MB-231cell lines; (F) MCF-7 cell lines, Control; (G) MWCNTs (10 µg/mL) treated MCF-7 cell lin es; (H) DTX (10 µg/mL) treated MCF-7 cell lines; (I) MWCNT-DTX (10 µg/mL) treated MCF-7 cell lines; (J) MWCNT-DTX-Piperine (10 µg/mL) treated MCF-7 cell lines. Comparisons were made between respective DTX and DTX-CNTs/DTX-CNTs-piperine treated groups, using student t-test. p-values *

Conjugation of Docetaxel with Multiwalled Carbon ... - ACS Publications

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