curcumin-nanocomposite for cancer therapy

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Efficient water soluble nanostructured ZnO grafted O-carboxymethyl chitosan/curcumin-nanocomposite for cancer therapy Laxmi Upadhyaya a,∗ , Jay Singh c,∗ , Vishnu Agarwal b , A.C. Pandey d , Shiv P. Verma e , Parimal Das e , R.P. Tewari a a

Department of Applied Mechanics, Motilal Nehru National Institute of Technology, Allahabad 211004, India Department of Biotechnology, Motilal Nehru National Institute of Technology, Allahabad 211004, India c Department of Applied Chemistry, Delhi Technological University, Shahbad Daulatpur, Main Bawana Road, Delhi 110 042, India d Nanotechnology Application Centre, University of Allahabad, Allahabad 211002, India e Centre for Genetic Disorders, Faculty of Science, Banaras Hindu University, Varanasi 221 005, India b

a r t i c l e

i n f o

Article history: Received 13 September 2014 Received in revised form 21 November 2014 Accepted 21 December 2014 Available online xxx Keywords: Carboxymethyl chitosan Drug delivery ZnO nanoparticles Cancer therapy

a b s t r a c t The present work deals with the synthesis of efficient water soluble O-carboxymethyl chitosan (O-CMCS) based nanocomposites (NCs) with nanostructured zinc oxide (n-ZnO) by ex-situ grafting method for the delivery of anticancer drug curcumin (Cr). The phase identification, morphology and thermal stability of prepared Cr/O-CMCS/n-ZnO NCs have been investigated by FT-IR, XRD, SEM, TEM and TGA/DTA techniques. The drug entrapment efficiency have found to be 74% and in vitro drug release study performed at 37 ◦ C at pH 4.5 and 7.4 indicated slow and controlled release in the initial phase and sustained in later phase. The MTT assay showed higher and preferential toxicity of the Cr/O-CMCS/n-ZnO NCs against cancer cells (MA104). The cellular uptake study by FACS revealed concentration dependent uptake of the NCs. The utilization of this Cr/O-CMCS/n-ZnO nanoformulation offers an efficient strategy and a novel promising soluble nanomatrix for anticancer therapy and other biomedical applications. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Cancer treatment still remains a big challenge to the medicine world as chemotherapies and radiotherapies are aggressive and poorly effective. These conventional anticancer therapies suffer from side effects such as fatigue, nausea, insomnia, delirium and vomiting which are common troubles for cancer patients [1,2]. For this reason, nanotherapeutics has attracted the attention of several researchers and scientists. In recent years, new development of nanomaterials, nanocomposites signify a promising area in the leading edge between the fields of materials science, life science, medical biology, including cancer therapy, have attracted significant attention. Various strategies in the drawing such as nanostructured materials, with unique size and shape surface morphology, govern the biodistribution, uptake, drug loading capacities, and properties for persistent or controlled release making nanostructured systems ideal and well suited for cancer therapy [3,4]. Nanomaterials has provided optimum size and

∗ Corresponding authors. Tel.: +91 1127871045; mobile: +91 9871765453. E-mail addresses: [email protected] (L. Upadhyaya), jay [email protected] (J. Singh).

surface characteristics are capable of enhancing therapeutic activity by prolonging drug half-life, improving solubility of hydrophobic drugs, reducing potential immunogenicity, and/or releasing drugs in a sustained or stimuli-triggered fashion. Thus, the toxic side effects and administration frequency of the drugs can be reduced [5]. Moreover, nanostructured materials can passively accumulate in specific tissues (e.g., tumors) through the enhanced permeability and retention (EPR) effect [6]. In particular, polymeric nanometer sized particles such as micelles, nanospheres, nanocapsules, polymerosomes, polyplexes, hydrogels, etc. have been predominantly in the attention as nano-carriers [7]. Polymeric nanoparticles carriers offer a large versatility in both structure and physiochemical properties due to a wide variety of available monomers. Drugs loading is accomplished by infusing the nanoparticles with drugs in aqueous phase resulting in highly controlled cage like or capsule conformations along with highly developed methodologies include trapping drugs by chemical cross-linking, modifying surface properties of nanoparticles, etc. [8,9]. Derivatives of chitosan like carboxymethyl chitosan (CMCS) have emerged as a promising functional biomaterial for efficient delivery of many of the highly potent hydrophobic anticancer drugs [10–14]. This is because CMCS can overcome some of the major limitations of chitosan like its poor aqueous solubility [15]. As

http://dx.doi.org/10.1016/j.procbio.2014.12.029 1359-5113/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Upadhyaya L, et al. Efficient water soluble nanostructured ZnO grafted O-carboxymethyl chitosan/curcumin-nanocomposite for cancer therapy. Process Biochem (2015), http://dx.doi.org/10.1016/j.procbio.2014.12.029

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compared to chitosan, CMCS has positive facets of increased water solubility, excellent biocompatibility [16], admirable biodegradability, high moisture retention ability [17], improved antioxidant property [18], enhanced antibacterial [19–21] and antifungal [22] activity and non-toxicity that might be suitable for efficient anticancer drug delivery applications [23]. The O-CMCS is an amphiprotic ether derivative of chitosan which contains COOH groups and NH2 groups in the molecule and has received considerable attention in the field of cancer therapy in the past decade. The preparation of O-CMCS by reacting chitosan with monochloroacetic acid in isopropyl alcohol as solvent at room temperature has been earlier reported in literature [21,24]. Despite these CMCS shows poor absorption, low stability, and short lifetime of drug into support matrix. Keeping this in view, many efforts have been made to develop suitable support matrices that may perhaps provide better environment for the efficient drug adsorption, improve solubility and high stability [25–27]. Polymer based nano drug carriers are growing interest that is an ability of certain metal oxide based nanomaterials to mediate anticancer effects on their own. Perelshtein et al. successfully prepared and characterized chitosan and chitosan–ZnO-based complex and studied their antibacterial activity without loss solubility, toxicity and adsorption phenomena [28]. One approach involves successful use of ZnO metal oxide NPs where ZnO NPs selectively induced apoptosis in cancer cells, which is likely to be mediated by reactive oxygen species via p53 pathway, through which most of the anticancer drugs trigger apoptosis [29]. Therefore, one of the reasons for using ZnO NPs in cancer treatment is that reduction of ZnO to the nanoscale has been shown to reveal toxic activity that is observed to preferentially target rapidly dividing cancerous cells [30,31], which is expected to serve as a foundation for developing novel cancer therapeutics. Curcumin [(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6heptadiene-3,5-dione] is a hydrophobic polyphenolic orangeyellow colored compound extracted from powdered rhizome of Curcuma longa (usually referred as turmeric or haldi). The excellent anti-carcinogenic [32], anti-HIV [33], anti-inflammatory [34], antioxidant [35] and antimicrobial [36] property has been well established by extensive research over the past 5 decades. Despite of its superior medicinal value and extremely high safety profile, the full exploitation of curcumin drug in the field of medicine, particularly cancer, still remains unexplored. The main factors that contribute to the low bioavailability and limited clinical efficacy of this highly potent anticancer drug are insolubility of curcumin in water at physiological pH, limited absorption, rapid metabolism, and excretion from the body [37]. Several attempts have been made till date to enhance the bioavailability of this drug by encapsulating in polymeric nanoparticles [38], solid lipid nanoparticles [39], polymeric micelles [40], biodegradable microspheres [41] and hydrogels [42,43]. The present study deals with the investigation of efficiency of O-CMCS/n-ZnO nanocomposite (NCs) in delivery of hydrophobic drug curcumin (Cr) for cancer treatment. We prepared O-CMCS coated ZnO fluid using ex situ grafting method into which anticancer drug Cr was loaded in order to solubilize and thereafter stabilize Cr in the O-CMCS/n-ZnO NCs. Addition of Cr to O-CMCS/n-ZnO not only caused agglomeration but also contributed to increased thermal stability as determined by SEM and thermal studies respectively. The entrapment efficiency was observed and which showed sustained and controlled release in vitro drug release. The drug loaded O-CMCS/n-ZnO NCs displayed significant cytotoxicity against cancer cell lines as evaluated by MTT assay. Overall, efficient water soluble O-CMCS/n-ZnO NCs have been found to be highly suitable for the sustained and controlled release of anticancer drug like Cr for cancer therapy and other biomedical applications.

2. Experimental 2.1. Materials Chitosan powder (molecular weight 100–150 kDa, DDA 80%) was a product of Sigma–Aldrich (USA). Sodium hydroxide (NaOH) and isopropyl alcohol were supplied by Qualigens. Zinc acetate (ZnAC2 ·2H2 O), 70% aqueous solution of glycolic acid, 25% aqueous solution of ammonia monochloroacetic acid and curcumin were purchased from Merck (India). Cell culture media like minimum essential medium (MEM) and Dulbecco’s modified eagle medium (DMEM) and MTT [3-(4,5-dimethylthiazole-2-yl)-2,5diphenyl tetrazolium] are purchased from Sigma–Aldrich (USA). All reagents were of analytical grade and used without any further purification.

2.2. Methods 2.2.1. Preparation of O-CMCS from chitosan The synthesis of O-CMCS was performed according to the method reported previously [24] with minor modifications. In brief, chitosan (2 g), NaOH (2.7 g) and solvent (20 mL) were mixed to form slurry and allowed to swell and alkalize at −20 ◦ C for 24 h. Then, a solution of monochloroacetic acid (3 g) dissolved in isopropanol (4 mL) was prepared. This solution of monochloroacetic acid was added to the slurry drop-wise at regular intervals followed by continuous stirring at 50 ◦ C for 6 h. The reaction was stopped by adding 70% ethanol to the reaction mixture. The solution was filtered, and washed with 70% ethanol four to five times and dehydrated with absolute alcohol. This primary product obtained was sodium salt of CMCS. This sample was immersed into 70% ethanol and then 32% HCl was added, mixed for 30 min and filtered in order to convert sodium salt of CMCS to CMCS. Finally, the product obtained was dissolved in deionized, water and dialyzed for 3 days to remove impurity and vacuum freeze dried. The degree of substitution was determined based on protocol reported earlier [24] and the dried sample was used for further characterization and NCs preparation.

2.2.2. Preparation of ZnO nanoparticles and O-CMCS/n-ZnO nanocomposite The synthesis of the ZnO nanoparticles was performed according to the method reported in our previous study [44]. Initially, we prepared 0.01 M aqueous solution of zinc acetate and subsequently 70% aqueous solution of glycolic acid was added into the above prepared solution with a molar ratio of metal salts/glycolic acid as 1:2. Further, 25% aqueous solution of ammonia was slowly added to the prepared solution while stirring until the pH of the solution reached up to ∼9. The resulting solution was evaporated to obtain transparent sol at 70–80 ◦ C for 4 h. To remove water from the sol, the transparent sol was heated once again at 70–80 ◦ C while being stirred. After complete evaporation of the water, the sol turned into a viscous transparent gel. Afterwards, the obtained gel was calcined at 600 ◦ C for 10 h to obtain nanocrystalline ZnO powder for further use. The O-CMCS coated ZnO fluid was synthesized using ex situ grafting method. For this, 15 mL of ZnO fluid (4 mg/mL) was added to 10 mL of aqueous O-CMCS solution (2 mg/mL) drop-wise and the reaction mixture was ultrasonically vibrated for 1 h. Then, the solution obtained was stirred for 24 h in order to get an O-CMCS-coated ZnO fluid. Finally, the solution was decanted, filtered off, washed with distilled deionized water 5 times in order to remove impurities and dried at 80 ◦ C for 4 h to obtain O-CMCS/n-ZnO nanocomposite (O-CMCS/n-ZnO NCs) powder.

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2.2.3. Preparation of curcumin-loaded O-CMCS/n-ZnO nanocomposite Firstly, a stock solution of curcumin was prepared in ethanol at a concentration of 2 mg/mL. Then an aqueous solution was prepared by dissolving 10 mg O-CMCS/n-ZnO nanocomposite in 20 mL Millipore water under stirring. To this solution, required volume of ethanolic curcumin was added drop-wise under continuous stirring for 48 h in a closed flask in order to load curcumin into O-CMCS/n-ZnO nanocomposite. Subsequently, the solution was stirred in open air for further 24 h to evaporate ethanol completely along with heating at 40–50 ◦ C to allow the penetration of Cur into O-CMCS/n-ZnO nanocomposites coat, resulting in the formation of curcumin/O-CMCS/n-ZnO nanocomposites (Cr/O-CMCS/n-ZnO NCs). Finally the suspension cooled and centrifuged at 20,000 rpm and washed three times with 20 mL portions of double distilled water, and re-suspended in phosphate buffer solution with pH = 7.4 until further use. Whenever required, these Cr/O-CMCS/n-ZnO NCs were dried in desiccators. Scheme 1 shows the pictorial representation for the preparation of O-CMCS/n-ZnO nanocomposites and (i) O-CMCS/n-ZnO/curcumin nanocomposite (ii). 2.2.4. Entrapment efficiency and loading efficiency To determine the entrapment efficiency of curcumin within the O-CMCS/n-ZnO NCs, the sample was pelletized by ultracentrifugation at 20,000 rpm at 4 ◦ C for 45 min from the aqueous medium containing free curcumin. The pellet obtained was washed thrice with distilled water, dispersed again in ethanol (2 mg pellet in 10 mL ethanol), vortexed well for 5 min and centrifuged at 20,000 rpm for 10 min. The amount of drug within the supernatant collected was quantified spectrophotometrically at 429 nm. Loading efficiency of the drug loaded NCs were also calculated with respect to the weight of the NCs obtained after centrifugation [45,46]. Entrapment efficiency (Amount of curcumin in the pellet) × 100% = Initial amount of curcumin

Loading efficiency =

(Total amount of curcumin) × 100% Yield of nanocomposite

(1)

(2)

2.2.5. In vitro drug release studies In vitro drug release profile of curcumin from drug loaded OCMCS/n-ZnO NCs were carried out by direct dispersion method for a period of a week at two different pH values (7.4 and 4.5) as described previously in literature [47]. The suspension of OCMCS/n-ZnO NCs was centrifuged at 20,000 rpm for 30 min and the pellet collected was dispersed again in 10 mL PBS (pH 7.4 and 4.5) at a final concentration of 0.2 mg/mL. The total amount of solution was divided into 20 eppendorf tubes with 0.5 mL each for a timedependent release study at time intervals of 1, 6, 12, 24, 48, 72, 96, 102 and 126 h. These tubes were incubated at 37 ◦ C under gentle shaking. At proper time intervals, curcumin in O-CMCS/n-ZnO NCs were first extracted in ethanol and then quantified spectrophotometrically [46]. Release was quantified as follows: Release (%) =

(Released curcumin) × 100% Total curcumin

(3)

2.2.6. Cell culture and cytotoxicity studies MA104 cell line was grown in Dulbecco’s modified Minimum Essential Medium (DMEM) (Sigma) supplemented with 10% fetal bovine serum (FBS) (Sigma) and antibiotics (Penicillin and streptomycin) (Sigma) in 96-well tissue culture plates. Cytotoxicity of the O-CMCS, pure curcumin, O-CMCS/n-ZnO NCs and Cr/OCMCS/n-ZnO NCs was studied via standard MTT assay, which is a

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colorimetric test based on the principle of selective ability of viable cells to reduce the tetrazolium component of MTT to purple-colored crystals. MA104 cells were seeded at a density of 10,000 cells/well into a 96-well plate and kept in CO2 incubator under standard culturing conditions. Five different concentrations of the O-CMCS, pure curcumin, O-CMCS/n-ZnO NCs and Cr/O-CMCS/n-ZnO NCs (1, 2, 3, 4 and 5 mg/mL) were prepared by dilution with the media. The cells were incubated with different concentrations of the NPs (100 ␮L) for a period of 24 h after reaching 90% confluency. Cells in media alone devoid of NPs/NCs (untreated cells) were used as a negative control (i.e., 100% viable) and the cells treated with toxin triton were used as positive control. Four hours before the end of experiment, MTT reagent was added in each well and kept at 37 ◦ C in CO2 incubator followed by 1 h incubation with solubilization buffer. Finally, the optical density of the solution was measured at a wavelength of 570 nm and cell viability (%) was determined. Triplicate samples were analyzed for each experiment. 2.2.7. Cell uptake studies 2.2.7.1. Cell uptake measurements by UV quantification. A comparative data of the amount of Cr/O-CMCS/n-ZnO NCs uptake by normal cells (L929) and cancer cells (MA104) was obtained by following the absorption spectrum of curcumin in the cell lysate. L929 and MA104 cell lines were incubated in 24-well plates, with a seeding density of 10,000 cells/well. Cr/O-CMCS/n-ZnO NCs (1 mg/mL in the medium) was added to each wells and incubated, after 90% confluency was reached. Then, the cells were washed well with PBS and trypsinized at definite time intervals of 1,6,12, 24 and 48 h incubation. Subsequently, the cells were lysed with ethanol under sonication, extracting the internalized particles and the lysate was centrifuged at 10,000 rpm for 10 min. Finally, supernatant containing ethanolic curcumin was quantified spectrophotometrically at 429 nm. 2.2.7.2. Cellular uptake by flow cytometry. 105 ACHN cells were seeded in 12-well plate and grown in DMEM supplemented with 10% fetal bovine serum (FBS) at 37◦ and 5% CO2 . After 24 h of growth cells were treated with 5 mg/mL Cr/O-CMCS/n-ZnO NCs and respective NCs as control. Intracellular curcumin fluorescence of 10,000 cells was analyzed by FACS Calibur (Beckton Dickinson) flow cytometer after excitation with a 488 nm argon laser. 2.2.7.3. Statistical analysis. All experimental values are presented as means ± SD. The FACS data were analyzed using Calibur (Beckton Dickinson), and then cytotoxicity test was used to analyze multiple differences between the groups by using Origin Pro software, version 7. P-value at less than 0.05 was considered statistically significant. 3. Characterization and measurements The crystalline nature and phase identification of ZnO, O-CMCS, O-CMCS/n-ZnO and Cr/O-CMCS/n-ZnO NCs were recorded on Xray diffractometer (Phillips X-pert model) with Cu K␣ radiation at ˚ Fourier transform infrared (FTIR) spectroscopy spectra  = 1.5406 A. of the prepared nanocomposite were recorded on a Perkin Elmer SPECTRUM GX-Raman spectrophotometer using KBr disk in the frequency range from 400 to 4000 cm−1 . The structural morphology and crystalline size of the synthesized ZnO nanocrystals is investigated by TEM measurements using Tecnai G2 S-Twin electron microscope. Surface morphology of the prepared nanocomposites was studied using SEM with JEOL JSM-5200 at 15 kV. The thermal behavior of O-CMCS, curcumin, O-CMCS/n-ZnO NCs and Cr/O-CMCS/n-ZnO NCs was studied through thermogravimetry and differential thermal analysis using a Perkin Elmer Pyris Diamond

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Scheme 1. Pictorial representation for O-CMCS/n-ZnO nanocomposites (i) and O-CMCS/n-ZnO/curcumin nanocomposite (ii).

TG/DTA/DSC 8.0 series instrument at a heating rate of 10 ◦ C under N2 atmosphere. 4. Results and discussion 4.1. FTIR and XRD FTIR spectra of n-ZnO NPs, O-CMCS, pure curcumin, O-CMCS/nZnO NCs and Cr/O-CMCS/n-ZnO NCs have been shown in Fig. 1A in the range of 400–4000 cm−1 . The FTIR spectrum of pure ZnO NPs (curve a) exhibits broad peak at 3358 cm−1 corresponding to O H stretching due to physically adsorbed water on ZnO surface. The two strong absorption bands at 471 cm−1 and 549 cm−1 are belonging to the stretching vibration mode and the torsional vibration mode of Zn O bonds in the tetrahedral sites and in the octahedral sites [48]. The IR spectrum of O-CMCS (curve b) characteristic peaks at 3431 cm−1 ( NH2 and OH stretching), at 2926 cm−1 ( CH stretching from aliphatic) and 1325 cm−1 ( C O stretching) were present. Also, the peaks at 1741 cm−1 ( COOH) and 1070–1136 cm−1 ( C O ) are the characteristics of O-CMCS. The FTIR spectrum of pure curcumin (curve c) shows characteristic absorption band at 3510 cm−1 , attributed to the phenolic O H stretching vibration. In addition to this, the absorption peaks 1609 (stretching vibrations of benzene ring), 1504 (C O and C C vibrations), 1430 (olefinic C H bending vibration) and 1274 cm−1 (aromatic C O stretching vibration) have clearly observed [49]. The IR spectrum of O-CMCS/n-ZnO NCs (curve d) shows different characteristic peaks of O-CMCS along with a sharp peaks at 424 and 574 cm−1 which corresponds to the presence of ZnO thereby confirming bonding between O-CMCS and ZnO forming nanocomposite [50]. The complexation of curcumin with O-CMCS in Cr/O-CMCS/n-ZnO NCs is confirmed by the shifts observed in the

wave numbers corresponding to the characteristic peaks of O-CMCS (curve e). When IR spectra of O-CMCS and Cr/O-CMCS/n-ZnO NCs are compared, peak shifts is observed from 3431 cm−1 to 3283 cm−1 and 1652 to 1647 cm−1 . This peak shift is due to electrostatic interaction between the curcumin and O-CMCS/n-ZnO NCs and it may be may be presence of curcumin in Cr/O-CMCS/ZnO NCs [51]. Fig. 1B shows the X-ray diffraction pattern of O-CMCS, n-ZnO NPs, O-CMCS/n-ZnO NCs and Cr/O-CMCS/n-ZnO NCs. The O-CMCS is typically a non-crystalline polymer and shows a broad diffraction peak appears at around 2 = 20◦ which indicates the amorphous nature (curve a). From the X-ray diffraction pattern, the positions and relative intensities of the reflection peak of the ZnO NPs (curve b), O-CMCS/n-ZnO NCs (curve c) and Cr/O-CMCS/n-ZnO NCs (curve d) are well agreed to each other. All the diffraction peaks corresponding to (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), planes are in agreement with typical wurtzite type structure of bulk hexagonal ZnO (JCPDS-card No. 36-1451) crystal [52]. Moreover, no peaks corresponding to any unknown phase could be observed in the spectra which further confirm the high purity of the obtained ZnO products. A broad peak with low intensity can be seen in the spectra ∼20 degree which might be associated with O-CMCS phase is due to both steric effect and intermolecular hydrogen bonds between ZnO particles and polymer chain [53]. The average crystallite size of ZnO crystal, O-CMCS/n-ZnO NCs and Cr/O-CMCS/n-ZnO NCs are determined by taking the full width at half maximum (FWHM) of four most intense diffraction peaks for each sample using the Debye Scherrer formula, d = 0.941 /ˇ cos , where  is the wavelength, ˇ is the FWHM of the diffracted peak, and  is the Bragg angle. The values of crystalline size thus obtained using FWHM of each peak for a particular nanocomposite is averaged out in order to determine the exact crystalline size of the sample. The average crystalline size of the ZnO crystal, O-CMCS/n-ZnO NCs and Cr/O-CMCS/n-ZnO NCs are

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Fig. 1. (A) FTIR spectra of (a) ZnO NPs, (b) O-CMCS, (c) pure curcumin, (d) O-CMCS/n-ZnO nanocomposites and (e) O-CMCS-ZnO/curcumin nanocomposite (B) X-ray diffraction pattern of (a) O-CMCS, (b) ZnO NPs, (C) O-CMCS/n-ZnO nanocomposites and (d) O-CMCS/n-ZnO/curcumin nanocomposite.

found to be 20, 22 and 27 nm respectively. During the formulation of curcumin, crystallinity of O-CMCS/n-ZnO NCs was well maintained with increasing the crystalline size (upto ∼5 nm). This result indicate that the successfully formulation of drug onto the surface of O-CMCS/n-ZnO NCs. 4.2. SEM and TEM analysis The surface morphologies of O-CMCS, ZnO NPs, O-CMCS/n-ZnO NCs and Cr/O-CMCS/ZnO NCs have been examined by using SEM as shown in Fig. 2a–d. A porous structure with abundant

interconnective open pores in the range of 1–3 ␮m could be clearly seen on all O-CMCS surface (image a). SEM image of prepared ZnO nanoparticles are homogenously distributed to adopted agglomerated spherical nanospheres (image b). The SEM image of O-CMCS/n-ZnO NCs shows the uniform granular nanoporous morphology with average grain size of about ∼40 nm which is attributed to the uniformly, homogeneous dispersion of ZnO nanoparticles in O-CMCS matrix (image c). It is suggested that O-CMCS can provide bridging of individual particles and suitable for the drug loading. After the incubation of curcumin onto the O-CMCS/n-ZnO matrix the granular morphology of O-CMCS/n-ZnO

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Fig. 2. (A) SEM images of (a) O-CMCS, (b) ZnO NPs, (C) O-CMCS/n-ZnO nanocomposites and (d) O-CMCS/n-ZnO/curcumin nanocomposite (B) TEM (image e) and HR-TEM image (g and h) and image f shows size distribution histograms and Lorentzian fits for sol–gel derived ZnO nanoparticles.

NCs changes into homogeneous globular porous morphology is due to the electrostatic interaction between O-CMCS/n-ZnO and curcumin. The images (image d) confirm that the particles are bigger in size with some extent of agglomeration is due to curcumin loading. The TEM micrographs of the ZnO nanoparticles are shown in Fig. 2e–h. The images clearly show that particles are smooth, spherical, agglomerate and evenly distributed nature of the particles [44]. The HR-TEM image (Fig. 2g and h) of prepared ZnO nanoparticles has shown the distances between the two lattice planes for nanostructured ZnO at around 0.28 nm, which correspond to the dspacing of the (1 0 0) crystal planes of the wurtzite ZnO. The average

particle size for the sol-gel derived ZnO nanoparticles was calculated to be ∼30 nm with help of the distribution curve fitted with Lorentzian function and shown in Fig. 2f. 4.3. Thermal analysis The thermal stability and thermal degradation behavior of the prepared O-CMCS, pure curcumin, O-CMCS/n-ZnO NCs and Cr/OCMCS/n-ZnO NCs were investigated by TGA study and the results are shown Fig. 3A (a–d). The TG curve clearly indicates that OCMCS degrades at a slower rate than pure curcumin and at 400 ◦ C,

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Fig. 3. (A) TGA and (B) DTA spectra of (a) O-CMCS, (b) pure curcumin, (c) O-CMCS/nZnO nanocomposites and (d) Cr/O-CMCS/n-ZnO nanocomposites.

while O-CMCS degrades to only 68%, curcumin is degraded to 60%. Despite of showing initial improved thermal stability (upto 300 ◦ C) in comparison to pure curcumin, results indicate that the nanocomposites prepared (O-CMCS/n-ZnO NCs, Cr/O-CMCS/n-ZnO NCs) show almost similar thermal degradation pattern as that of pure curcumin. As per DTA (as shown in Fig. 3B (a-d)), an endothermic peak of crystalline curcumin was observed at 185 ◦ C (curve b), which completely disappeared in Cr/O-CMCS/n-ZnO NCs (curve d) thereby confirming the high amorphous nature of the nanoformulation and consequent better stability. Speaking specifically, the complexation of a drug and polymer causes the absence/shifting of the endothermic peak which indicates a change in the crystal lattice, melting, boiling, or sublimation points [49]. Also, the more is amorphous nature of the therapeutic system; the ahead of higher will be its drug delivery efficiency [49]. 4.4. Entrapment efficiency and in vitro drug release studies Entrapment and loading efficiency of curcumin in O-CMCS/nZnO NCs have been found to be 74% and 43%, respectively. The results indicated that the amount of the O-CMCS/n-ZnO matrix and curcumin had critical effects on the drug absorption. Higher amount of O-CMCS/n-ZnO NCs facilitate more loading and entrapment, but resulted in large sized particles. However at higher

Fig. 4. (A) Drug release profile of curcumin from O-CMCS-n-ZnO/curcumin nanocomposite in pH 4.4 and 7.5 at 37 ◦ C (value were indicate the error bar, SD ±3). (B) Water solubility of curcumin (a), O-CMCS/n-ZnO nanocomposite (b) and Cr/O-CMCS/n-ZnO nanocomposites (c).

curcumin concentration entrapment efficiency have been reduced where the drug tends to precipitate. As considering all these parameter, concentration of O-CMCS/n-ZnO NCs and curcumin have been optimized so as to give better entrapment, loading efficiency and desired size. Fig. 4A shows the in vitro drug release profile of curcumin loaded in Cr/O-CMCS/n-ZnO NCs at two different pH values (4.5 and 7.4) [54]. The release of the drug is slow and controlled in the initial phase which becomes sustained finally. The drugrelease curve (Fig. 4A) clearly shows that at the 10-h there is a 23% and 28% curcumin release from O-CMCS/n-ZnO matrix which reached at 35% and 48% at pH 7.4 and 4.5 respectively after 6 days. The release is faster in acidic pH than neutral pH which can be attributed to protonation of residual amine groups in O-CMCS of the Cr/O-CMCS/n-ZnO NCs at lower pH, creating a repulsive force between the adjacent positive charges thereby causing the polymer to swell. Hence more amount of drug will be diffused out. On contrary, limited swelling in basic medium inhibits diffusion of drugs at a faster rate as compared to faster drug diffusion rate in acidic

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Table 1 The solubility of O-CMCS, O-CMCS/n-ZnO NCs, Cr/O-CMCS/ZnO NCs and pure curcumin in various solvents. b

Solvent

Acetic acid (1%) Chloroform DMSO Dichloromethane 1,4-Dioxane Acetone Diethyl ether Xylene Hexane Benzyl alcohol Heptanol Isoamyl alcohol Ethyl alcohol Methanol Water a b

a

O-CMCS

VS IS IS IS IS IS IS IS IS SS SS SS SS SS SS

O-CMCS/n-ZnO nanocomposite

Curcumin/O-CMCS/n-ZnO nanocomposite

Curcumin

S SS SS SS SS SS SS SS SS S S S S S S

S S S SS SS S S SS SS S S S S S VS

SS VS VS S S VS VS S S SS S S VS VS IS

O-CMCS: carboxymethyl chitosan, n-ZnO: nanostructured zinc oxide. VS: very soluble, S: soluble, SS: slightly soluble, IS: insoluble.

medium [55]. The observed sustained release in the later phase has significant as controlled drug release is desirable in the field of cancer therapy thereby indicating that the nanoformulation prepared is a useful controlled anticancer drug delivery system. Table 1 shows the solubility of O-CMCS, O-CMCS/n-ZnO NCs, Cr/O-CMCS/ZnO NCs and pure curcumin in various solvents. Fig. 4B demonstrates that while curcumin as such is insoluble in water, curcumin loading into O-CMCS/n-ZnO NCs enhanced the drug solubility in water. As a result of which loading of curcumin into O-CMCS/n-ZnO NCs enhanced the aqueous solubility of curcumin.

4.5. In vitro cytotoxicity via MTT assay The potential toxicity of O-CMCS, O-CMCS/n-ZnO NCs, Cr/OCMCS/n-ZnO NCs and pure curcumin, has been studied by using normal cell (L929) and cancer cell (MA104) lines via MTT assay as shown in Fig. 5A and B respectively. When normal cells (L929) exposed to O-CMCS displayed higher cell viability percentage while cell viability reduced against O-CMCS/n-ZnO NCs, Cr/OCMCS/n-ZnO NCs and pure curcumin in the concentration range of 1–3 mg/mL. Almost 80% of the cells were viable displaying the non-toxicity of O-CMCS, O-CMCS/n-ZnO NCs, Cr/O-CMCS/n-ZnO NCs and curcumin against normal cells. Similarly, MA104 cells when exposed to O-CMCS showed no toxicity while their viability reduced when exposed to O-CMCS/n-ZnO NCs, Cr/O-CMCS/n-ZnO NCs and pure curcumin in the concentration range of 1–3 mg/mL. In general, the cytotoxicity assay showed comparatively higher toxicity of the Cr/O-CMCS/n-ZnO NCs against MA104 cancer cell lines than normal cells (L929). Both, the preferential toxicity of the nanoformulation prepared (Cr/O-CMCS/n-ZnO NCs) against cancer cells and higher toxicity of O-CMCS/n-ZnO NCs and Cr/O-CMCS/nZnO NCs than O-CMCS can be attributed to the presence of ZnO NPs in the hybrid nanocomposites formation. In particular, ZnO when reduced from bulk to nanoscale has been shown to exhibit inherent preferential cytotoxicity against cancer cells in vitro [30,31]. Therefore, MTT results showed that the Cr/O-CMCS/n-ZnO NCs showed significant toxicity due to which cell viability reduced to 24% at 3 mg/mL concentration thereby confirming its anticancer effects. The comparable anticancer activity of Cr/O-CMCS/n-ZnO NCs and curcumin revealed that curcumin retained its anticancer activity even after being loaded into polymer matrix and presence of ZnO NPs enhances the anticancer effects. The toxicity images of cancer cells (MA104) treated with pure curcumin (a) Cr/O-CMCS/n-ZnO NCs (b) and O-CMCS (c) are shown in Fig. 6.

Fig. 5. Cell viability of (A) L929 normal cell (B) MA104 cancer cell for (a) O-CMCS, (b) O-CMCS-ZnO nanocomposites, (c) O-CMCS-ZnO/curcumin nanocomposite and (d) pure curcumin Note: *significantly different (<0.05) from the O-CMCS; # significantly different from O-CMCS-ZnO nanocomposites; ˆsignificantly O-CMCS-ZnO/curcumin nanocomposite and $ significantly different from pure curcumin.

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Fig. 6. Toxicity images of cancer cells (MA104) treated with (a) pure curcumin (b) Cr/O-CMCS/n-ZnO NCs and (c) O-CMCS

4.6. In vitro cell uptake study 4.6.1. Cell uptake study by UV quantification The cellular uptake study of Cr/O-CMCS/ZnO NCs by normal (L929) and cancer (MA104) cells are carried out by using ethanol extraction method. In this method the released curcumin within

Fig. 7. UV absorbance spectrum of ethanolic O-CMCS/n-ZnO/curcumin nanocomposite from (A) normal cell and (B) MA104 cancer cell in different time (1–48 h) interval. Inset shows the absorption spectra with respect to time.

the cells is quantified spectrophotometrically. The absorption spectra of ethanolic curcumin extracted from L929 and MA104 respectively is shown in Fig. 7A and B. The absorption spectra indicate an increased release of drug within MA104 cells as compared

Fig. 8. The cellular uptake profile of Cr/O-CMCS/n-ZnO NCs by FACS studies in MA104 cancer cells.

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to L929 cells with increased time intervals. These results confirm more cellular internalization of Cr/O-CMCS/n-ZnO NCs within cancer cells as compared to normal cells [56]. 4.6.2. Cell uptake study by FACS analysis The cellular uptake study of Cr/O-CMCS/n-ZnO NCs by ACHN cells has been performed by flow cytometry. The cellular uptake profile of Cr/O-CMCS/n-ZnO NCs by ACHN cells is shown in Fig. 8. It shows an increase in the uptake of the nanoformulation prepared (Cr/O-CMCS/n-ZnO NCs) by the cancer cell line (ACHN cells) with the increase in concentrations of Cr/O-CMCS/ZnO NCs. Hence the results indicate that the uptake of the nanoformulation synthesized is concentration dependent. The efficient nanoformulated platform opens the new perspectives for in vitro drug release and targeted drug delivery. 5. Conclusions In summary, the O-CMCS/n-ZnO NCs were prepared by ex situ grafting method where ZnO nanoparticles have been synthesized by sol–gel method. Curcumin was effectively loaded into O-CMCS/n-ZnO NCs and characterized by XRD and SEM and TEM analysis. The FTIR spectra of O-CMCS/n-ZnO NCs confirmed the bonding between ZnO and O-CMCS and complexation of curcumin with O-CMCS in Cr/O-CMCS/n-ZnO NCs. The thermal behavior of OCMCS/n-ZnO NCs was found to be similar to pure curcumin. In vitro drug release was observed to be slow and controlled in the initial phase while sustained in the later phase. Preferential and higher toxicity of Cr/O-CMCS/n-ZnO NCs against MA104 cancer cell lines than normal cells (L929) was indicated by MTT assay. Higher cellular internalization of Cr/O-CMCS/n-ZnO NCs within cancer cells (MA104 cell line) as compared to normal cells (L929 cell line) was confirmed by cellular uptake study by UV quantification while concentration dependent cell uptake of the nanoformulation prepared (Cr/O-CMCS/ZnO NCs) was indicated by the results of cell uptake study by FACS analysis. The results clearly suggest that O-CMCS/nZnO NCs is a promising platform for anticancer therapy and other biomedical applications. Acknowledgements The authors would like to thank Ministry of Human Resource Development, Govt. of India for providing fellowship for research. We also want to acknowledge the Director, Motilal Nehru National Institute of Technology, Allahabad, India for providing other necessary facilities for research work. JS is kindly acknowledged to Department of Science and Technology, New Delhi, India for awarding the INSPIRE FACULTY AWARD [IFA-13-CH-105]. References [1] Hrushesky WJM, Grutsch J, Wood P, Xiaoming Y, Oh E-Y, Ansell C, et al. Circadian clock manipulation for cancer prevention and control and the relief of cancer symptoms. Integr Cancer Ther 2009;8:387–97. [2] Savard J, Morin CM. Insomnia in the context of cancer: a review of a neglected problem. J Clin Oncol 2001;9(3):895–908. [3] Petros RA, DeSimone JM. Strategies in the design of nanoparticles for therapeutic applications. Nat Rev Drug Discov 2010;9:615–27. [4] Farokhzad OC, Langer R. Impact of nanotechnology on drug delivery. ACS Nano 2009;3:16–20. [5] Shi J, Votruba AR, Farokhzad OC, Langer R. Nanotechnology in drug delivery and tissue engineering: from discovery to applications. Nano Lett 2010;10:3223–30. [6] Matsumura Y, Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent SMANCS. Cancer Res 1986;46:6387–92. [7] Liechty WB, Peppas NA. Expert opinion: responsive polymer nanoparticles in cancer therapy. Eur J Pharm Biopharm 2012;80:241–6. [8] Bajpai AK, Shukla SK, Bhanu S, Kankane S. Responsive polymers in controlled drug delivery. Prog Polym Sci 2008;33:1088–118.

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