http://informahealthcare.com/mnc ISSN: 0265-2048 (print), 1464-5246 (electronic) J Microencapsul, Early Online: 1–11 ! 2014 Informa UK Ltd. DOI: 10.3109/02652048.2014.940015

RESEARCH ARTICLE

Carboxymethyl starch-chitosan-coated iron oxide magnetic nanoparticles for controlled delivery of isoniazid Journal of Microencapsulation Downloaded from informahealthcare.com by University Of South Australia on 08/11/14 For personal use only.

Chinmayee Saikia1, Anowar Hussain2, Anand Ramteke2, Hemanta K. Sharma3, and Tarun K. Maji1 1

Department of Chemical Sciences, Tezpur University, Sonitpur, Assam, India, 2Department of Molecular Biology and Biotechnology, Tezpur University, Sonitpur, Assam, India, and 3Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh, Assam, India

Abstract

Keywords

Context: The coating material of magnetic nanoparticles plays a great role in drug delivery application. The coatings not only increase the stability of the nanoparticles but also improve the drug release pattern, biocompatibility and mucoadhesivity. Objective: Montmorillonite (MMT) containing magnetic iron oxide nanoparticles coated with polyelectrolyte complex (PEC) of carboxymethyl starch-chitosan were prepared for controlled release applications. Method: The PEC-coated nanoparticles were characterised by Fourier Transmission Infra-red spectroscopy and X-ray diffraction, scanning electron microscope, transmission electron microscope, and dynamic light scattering. Cytotoxicity study was performed by MTT assay analysis. Mucoadhesivity test was performed by using in vitro wash off and ex vivo method. Result: The coating of PEC showed good stability, biocompatibility and mucoadhesivity of the iron oxide magnetic nanoparticles. MMT addition enhanced the swelling, drug loading and release and also the cytotoxicity and mucoadhesivity of the nanoparticles. Conclusion: This study revealed that the MMT incorporated PEC of CMS-CS can be effectively used for coating of iron oxide nanoparticles.

Drug delivery, iron oxide, isoniazid, MMT, polyelectrolyte complex History Received 24 February 2014 Revised 24 June 2014 Accepted 25 June 2014 Published online 30 July 2014

Abbreviations: PEC: polyelectrolyte complex; CMS: carboxymethyl starch; CS: chitosan

Introduction Magnetite nanoparticles have drawn much interest in many areas recently, mainly in biomedical applications such as magnetic resonance imaging (MRI), tissue engineering, hyperthermia and drug delivery (Latham and Williams, 2008; Suna et al., 2008). Due to their biocompatibility, low toxicity and superparamagnetic character, magnetite nanoparticles are proved to be potential candidate for targeted drug delivery (Selim et al., 2006). However, the main disadvantage related to magnetic nanoparticles is their tendency for agglomeration, due to strong magnetic dipole–dipole attractions between particles, during synthesis process (Kim et al., 2001). Hence, the key feature in the preparation of magnetic nanoparticles is to prevent agglomeration by using surface coating agents like different types of water soluble polymers, e.g. polyethyleneglycol (PEG), dextran, chitosan (CS), starch (Neuberger et al., 2005). In the recent years, developing of newer strategies for optimising the surface of these nanoparticles becomes a challenge for the researchers. Polyelectrolyte complexes (PEC) are the association complexes formed between two oppositely charged polymers. These complexes have unique physicochemical properties of different polymers with high biocompatibility. Such complex may possess unique properties that are different from those of individual

Address for correspondence: Tarun K. Maji, Department of Chemical Sciences, Tezpur University, Sonitpur 784028, Assam, India. Tel: +91 3712 267007; Extn: 5053. Fax: +91 3712 267005. E-mail: [email protected]

components. PECs are found to possess great potential in the design of novel drug delivery system. PEC can be synthesised by simple blending of two oppositely charged polymers in aqueous solution (Prajapati and Sawant, 2009). Carboxymethyl starch (CMS), an ether derivative of starch, has been experimentally used as an efficient drug carrier (Nabais et al., 2007). It is an important modified starch having unique properties owing to the presence of negatively charged functional groups (CH2COO). CS, a naturally occurring cationic polysaccharide, has also been greatly explored for its potential in drug delivery technology. It has much attractiveness because of its good biocompatibility, anti-microbial–anti-fungal property and enhanced mucoadhesivity due to its cationic nature (Rajan and Raj, 2013). CS is a good dispersant for iron oxide nanoparticles because of its interaction with metal ions and metal oxide nanoparticles through the primary amines. Although both CMS and CS are a very promising biopolymer for use as coating material for magnetic nanoparticles, they show limited capacity for controlling drug release due to its fast dissolution in the stomach. Hence, PEC of CMS and CS has been established to surmount this disadvantage and to provide the required physicochemical properties for the design of specific magnetic nanoparticle based drug delivery system. PEC can provide better controlled release of drug than either material alone. Further, varieties of cross-linking agents are generally employed to enhance the efficiency of controlled release system. Montmorillonite (MMT) clay is smectite clay having silica tetrahedral sheets layered between alumina octahedral sheets. Due to the special chemical structure and large surface area, it imparts

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good adsorbability, enhanced drug loading and controlled drug release capacity, along with mucoadhesivity property (Wang et al., 2008). MMT-incorporated PEC of CMS-CS will provide excellent drug delivery properties. Isoniazid is a widely used, potent anti-tuberculosis drug effective against Mycobacterium tuberculosis (Bhowmik et al., 2009). The efficient treatment of this disease is limited by the toxicity of the drugs, the degradation of drugs before reaching required zones in the body, and low permeability of cell membranes to the drugs (Devi and Maji, 2009). Introduction of CMS-CS-coated iron oxide magnetic nanoparticles as a carrier matrix will facilitate controlled delivery of isoniazid and prevent the premature degradation of drug molecules upon administration within the body. Several reports on both magnetic nanoparticles and PEC for drug delivery applications are available in literature. However, not much is known regarding the combination of both and the use of MMT in drug delivery system. In case of drug delivery application, the surface coating is the main key factor for magnetic nanoparticles to attain stability, biocompatibility, requisite size, magnetic properties and optimum drug release properties. In this study attempts have been made to use PEC of CMS–CS as the surface coating agent. As MMT has established as a useful ingredient for controlling drug release from polymer matrix, effort have also been made to incorporate it in the PEC complex and to study the effect of variation of MMT concentration on various properties of the complex-coated nanoparticles.

meantime, solution of 0.5 g of FeCl36H2O and 0.25 g of FeCl24H2O was prepared in 25 mL of water and added to the above polymer solution. Again, in a beaker, MMT (0.005– 0.025 g) (1, 3 and 5% w/w, w.r.t. polymer) was swelled in 25 mL of water for 24 h. It was then stirred vigorously by mechanical stirrer for 48 h and sonicated for 30 min. The swelled MMT solution was slowly added to the iron containing polymer solution under stirring condition. To this, 0.01 g of isoniazid along with 3% Tween 80 (v/w, w.r.t. polymer) was added slowly and sonicated for 15 min. Now, a solution of 1.5 M NaOH was added dropwise to this solution under vigorous stirring at 60  C for approximately 2 h until black precipitate occurred. The precipitate thus obtained was washed with water until the pH became less than 8.5. Now, the temperature of the reaction mixture containing the nanoparticles was brought down to 10  C. The obtained nanoparticles were then crosslinked by slow addition of 5% glutaraldehyde (v/w, w.r.t. polymer) and the temperature was gradually raised to 45  C. The reaction was continued for 1 h. The mixture was then cooled to room temperature. The product was filtered, washed and dried under vacuum. A series of six samples as represented in Table 1 were prepared for the present study. Calculation of process yield Process yield was calculated using the following equation as described in the literature (Gupta and Kumar, 2000). Process yield ð%Þ ¼

Materials and methods

Weight of nanoparticles ðweight of drug þ weight of polymerÞ  100:

Materials Starch, isopropanol, iron (II) chloride tetrahydrate, iron (III) hexahydrate, tween 80, sodium monochloroacetate, glutaraldehyde, sodium hydroxide pellets were purchased from Merck, India. Low molecular weight CS, MMT K-10, isoniazid and histopaque 1077 and [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] (MTT) were obtained from Sigma Aldrich, Germany. RPMI 1640 and fetal bovine serum (FBS) were procured from HiMedia Laboratories (Mumbai, India). The rest of the chemicals were of analytical grade and used as such received. Preparation of drug-loaded PEC-coated magnetic iron oxide nanoparticles CMS was prepared using the procedure as reported in the literature (Saboktakin et al., 2011). Now, CMS and CS solutions were prepared by dissolving 0.25 g of CMS in 25 mL of water and 0.25 g of CS in 25 mL of 0.5% (v/v) acetic acid solution. These solutions were mixed together with continuous stirring. In the

Calibration curve of Isoniazid A calibration curve is necessary to estimate the release rate of drug from nanoparticles in a suitable solvent medium. A known concentration of isoniazid was scanned (in double-distilled water) in the range 200–400 nm by using UV–Visible spectrophotometer (UV-2001Hitachi, Tokyo, Japan). For isoniazid having concentration in the range 0.001–0.01 g/100 mL, a prominent peak at 262 nm was observed. The absorbance values at 262 nm obtained with the respective concentration were recorded and plotted. With the help of this calibration curve, the unknown concentration of isoniazid was obtained by knowing the absorbance value. Determination of isoniazid loading and encapsulation efficiency of the nanoparticles To determine the encapsulation efficiency and loading capacity, nanoparticles with the different formulation were centrifuged at room temperature for 30 min, and the amount of free Isoniazid

Table 1. Recipes for the composition of different isoniazid-loaded CMS-CS PEC-coated iron oxide nanoparticles and effect of variation of MMT concentration on the different properties of the nanoparticles. Average diameter (nm)c Sample code* NP/M0/GA5 NP/M1/GA5 NP/M3/GA5 NP/M5/GA5 NP/M3/GA0

MMT% (w/v) w.r.t. polymer (g in 50 mL H2O) 0 1 3 5 3

(0.000) (0.005) (0.015) (0.025) (0.015)

GA% (v/v) w.r.t. polymer (amount in mL) 5 5 5 5 0

(0.025) (0.025) (0.025) (0.025) (0.000)

Yield of nanoparticles (%)

Encapsulation efficiencya (%)

Loading efficiencyb (%)

88.40 ± 0.05 90.80 ± 0.08 91.00 ± 0.03 91.50 ± 0.01 91.50 ± 0.05

65.40 ± 0.01 72.70 ± 0.04 74.90 ± 0.01 68.90 ± 0.05 70.60 ± 0.01

20.60 ± 0.02 26.30 ± 0.03 28.10 ± 0.02 22.10 ± 0.03 24.40 ± 0.05

TEM 143 150 165 147 167

(±3) (±2) (±1) (±1) (±2)

DLS 242 241 247 232 219

(±14) (±13) (±10) (±11) (±11)

Notes: CMS: 0.25 g, CS: 0.25 g, FeCl36H2O: 0.05 g, FeCl24H2O: 0.25 g, Isoniazid: 0.01 g, Tween 80: 0.015 mL, H2O: 100 mL. *In the sample code, nanoparticles, MMT and glutaraldehyde are represented by ‘NP’, ‘M’ and ‘GA’, respectively. *a, b, c, d: The value represents average of five readings, standard deviation in parenthesis.

Zeta potentiald (mV) 30.34 40.22 42.12 39.32 33.16

(±0.03) (±0.01) (±0.04) (±0.03) (±0.01)

DOI: 10.3109/02652048.2014.940015

was determined in clear supernatant by UV spectrophotometer at 262 nm. Encapsulation efficiency (EE) and loading efficiency (LE) were calculated using the following formulae (Tripathi et al., 2010): Encapsulation efficiency ðEEÞð%Þ ¼

ðTotal amount of drug  Free amount of drugÞ  100 Total amount of drug

Loading efficiency ðLEÞð%Þ ¼

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ðTotal amount of drug  Free amount of drugÞ  100 Weight of dry nanoparticles Characterisation FTIR spectra of nanoparticles were taken in Nicolet (model Impact-410) spectrophotometer in the range of 4000–400 cm1. X-ray diffraction (XRD) study was carried out in a Rigaku X-ray diffractometer (Miniflex, Tokyo, Japan) using CuKa ( ¼ 0.154 nm) radiation at a scanning rate of 1 /min with an angle ranging from 2 to 70 of 2y. The magnetic properties of the nanoparticles were studied by using magnetic susceptibility analyser (Model MS2WF, Bartington, UK) instrument at ramp rate of 5  C/min in temperature range of 30 –600  C. Thermal properties of the nanoparticles were evaluated using a thermogravimetric analyser (Model TGA-50, Shimadzu, Singapore) instrument at a heating rate of 5  C/min upto 500  C under nitrogen atmosphere. The surface morphologies of the nanoparticles were investigated by using JEOL JSM-6390LV Scanning Electron Microscope (SEM) at an accelerated voltage of 5–10 KV. The particle size was examined by using JEOL JEM-2100 Transmission Electron Microscope (TEM) at an accelerated voltage of 100 KV and the Particle size distribution was determined by dynamic light scattering (DLS) analyser (model DLS-Nano ZS, Zetasizer, Nanoseries, Malvern Instruments, UK). Swelling studies Nanoparticles (0.1 g) were immersed in phosphate buffer (pH 7.4) for different time periods. After a definite time period, the nanoparticles were removed, blotted with filter paper, and changes in weight were measured and recorded. Swelling percentage was then determined from the following formula (Devi and Maji, 2010):   ðw2  w1 Þ  100 Swelling ð%Þ ¼ w1 where ‘‘w1’’ is the initial weight of nanoparticles before swelling and ‘‘w2’’ is the final weight of nanoparticles after swelling for a predetermined time ‘‘t’’. In vitro drug release studies In order to study the release profile of the isoniazid-loaded nanoparticles, dried drug-loaded test samples were immersed in a solution of different pH namely 1.2 and 7.4 and stirred continuously. At scheduled time interval, 5 mL solution was withdrawn, filtered and assayed spectrophotometrically at 262 nm by using UV–Visible spectrophotometer for the determination of cumulative amount of drug release upto a time t. Each determination was carried out in triplicate. To maintain a constant volume, 5 mL of the solution having same pH was returned to the container (Cassano et al., 2012). Identical experiments were performed in the presence of magnetic field (0.2T and 0.5 T) using an electromagnet.

Coating of iron oxide nanoparticles in drug delivery

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Isolation of lymphocytes, culture and treatment Cytotoxicity of the drugs, components and different formulations were done using Isolated Human Lymphocytes. The work was approved by Institutional Ethical Committee. Anti-coagulated human blood, collected voluntarily, was diluted with phosphate buffer saline (PBS) (1:1, v/v), layered 6 mL into 6 mL Histopaque (1.077 gm/mL), centrifuged at 400  g for 30 min, and lymphocytes were isolated from the buffy layer. Isolated lymphocytes were then washed with 2 mL PBS and 2 mL RPMI-1640 media separately through centrifugation for 10 min at 250  g (Thorsby and Bratlie, 1970; Winchester and Ross, 1976). Pelleted lymphocytes were then suspended in RPMI, and viability was checked by Trypan blue exclusion method using haemocytometer (Lecho´n et al., 1992). Lymphocytes with viability more than 90% were used for subsequent study. Aliquots of 200 mL of isolated cells were cultured in RPMI supplemented with 10% heat inactivated FBS. Initially, cells were maintained for 4 h in RPMI without FBS at 37  C in 5% CO2 in an incubator. Cells were then treated as per experimental requirements and maintained in the presence of FBS for 8 h. Cytotoxicity experiments Cytotoxicity assay was performed by measuring the viability of cells according to the method as described by Denizot and Lang (1986). The key component (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) (MTT) is yellowish in colour and mitochondrial dehydrogenase of viable cells cleave the tetrazolium ring, yielding purple insoluble formazan crystals which were dissolved in suitable solvent. In this report, dimethyl sulfoxide (DMSO) is used as the control solvent. The resulting purple solution was spectrophotometrically measured. An increase or decrease in cell number resulted in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by the test material. Briefly, after treatments, cells were treated with 10% of MTT for 2 h followed by dissolving the formazan crystals in solvent and measuring the absorbance of solution at 570 nm. The absorbance of control cells at 6, 12 and 24 h were separately set as 100% viability and the values of treated cells were calculated as percentage of control. Mucoadhesive study In vitro wash-off test The mucoadhesive property of the nanoparticles was evaluated by an in vitro adhesion test method known as wash-off test method (Lehr et al., 1990). Freshly excised pieces of goat intestinal mucosa (5  5 cm) were mounted with mucous side exposed on to glass slides with cotton thread. About 50 nanoparticles were spread on to each prepared glass slide and immediately thereafter the slides were hung to USP tablet disintegration test apparatus (Tab. Machines, Mumbai, India). When the test apparatus was operated, the sample was subjected to slow up and down movement in the test fluid at 37  C contained in a 1 L vessel of the apparatus. Readings were taken at an interval of 30 min up to 5 h by stopping the machine and counting the number of nanoparticles still adhering to mucosal surface. The test was performed at intestinal (pH 7.4) and simulated gastric fluid (pH 1.2) condition. Ex vivo mucoadhesive test In this method, the force required to separate bio-adhesive sample from freshly excised goat intestine was determined (Sharma et al., 2013). Keeping the mucosal side out, the intestine was secured on to each glass vial using nylon thread. The diameter of each exposed mucosal membrane was 2 cm. The vials with the nasal

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tissue were kept at 37  C for 10 min. To the exposed tissue on this vial, a constant amount of nanoparticle was applied. The height of the vial was adjusted so that the nanoparticles could adhere to the mucosal tissues of both vials. Water was added at a constant rate to the pan on the other side of the modified balance until the two vials were separated. The weight of water showed the weight required for displacement. The adhesive force was calculated using the following equation. Detachment stress (dyne cm2) ¼ mg/Awhere ‘‘m’’ is the mass (g) required to detach the membrane, ‘‘g’’ is acceleration due to gravity taken as 980 cm sec2 and ‘‘A’’ is the area of tissue exposed which is equal to  r2 (r is the radius of the exposed mucosal membrane). Journal of Microencapsulation Downloaded from informahealthcare.com by University Of South Australia on 08/11/14 For personal use only.

Statistical analysis All the data were expressed as means ± SD. Results were statistically analysed by student’s t-test for significance difference between group mean using GraphPad software. The significant difference between the experimental and the control group was set at different levels as p50.05, p50.01 and p50.001.

Results and discussions Effect of variation of MMT on different properties of CMS-CS PEC-coated iron oxide nanoparticles Table 1 shows the influence of glutaraldehyde and variation of MMT on different properties of the CMS-CS-coated iron oxide nanoparticles. It was observed that addition of MMT did not have any significant effect on yield (%) of the nanoparticles. But both encapsulation and loading efficiency were found to enhance upto a certain concentration of MMT, beyond that the value decreased. This could be attributed to the unique physical structure of MMT. The layered silicate structure of MMT provided large specific area and network structure in the polymer matrix which could load the drug more effectively than the nanoparticles without MMT (Zheng et al., 2011). Hence, both encapsulation and loading efficiency were found to increase with the increase in MMT concentration. However, beyond a certain MMT concentration, the polymeric network structure might be collapsed due to which loading capacity of the nanoparticles decreased (Hua et al., 2010). Addition of crosslinker also facilitated the encapsulation and loading efficiency of the nanoparticles by forming complex polymer network structure. Therefore, the encapsulation as well as drug loading efficiency of glutaraldehyde free nanoparticles was found less than the glutaraldehyde added nanoparticles. A schematic representation of the formation of glutaraldehyde cross-linked nanoparticles is shown in Figure 1. Figure 1. Schematic representation of the formation of the glutaraldehyde cross-linked polyelectrolyte complex of CMS-CS-coated magnetic Fe3O4 nanoparticles.

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The average diameter of the nanoparticles was obtained in the range of 143–167 nm as shown by TEM study. Again, the particle size distribution was estimated from DLS data. From the DLS data, particle size distribution was found to be in the range of 219–247 nm. The difference in diameter between the TEM measurement and the DLS measurement might be due to swelling of the coated nanoparticles in solution. The addition of MMT did not bring about any significant difference in particle size. Stability of the nanoparticles could be confirmed by their zeta potential values. In our study, zeta potential values for the nanoparticles were found in the range of 30.34 to 42.12 mV indicating good stability of the nanoparticles. Nanoparticles containing MMT showed more negative surface charge compared to MMT free nanoparticles. This could be due to negative charges carried by MMT surface (Galindo and Viseras, 2004). Nanoparticles with glutaraldehyde showed higher zeta potential than the nanoparticles without glutaraldehyde indicating their greater stability due to crosslinking. The crosslinking of the polymer coating of the magnetic nanoparticles fueled the stability of the nanoparticles and also maintained their size. Fourier transmission infra-red spectroscopy study In the spectrum of CMS (curve a), a broad peak was appeared at 3450 cm1 due to O–H stretching vibrations of CMS. The peaks at 2880, 1750, 1620, and 1420 cm1 were assigned for C–H stretching vibration and C¼O and COO bonds in carboxylic salts in CMS structure. In the spectrum of CS (curve b), the broad absorption band appeared in the range 3200–3480 cm1 was due to the hydrogen-bonded OH stretching and NH2 asymmetric stretching. The characteristic peaks of amide I and amide II were appeared at 1640 cm1 (C¼O stretching) and 1560 cm1 (N–H in plane deformation coupled with CN stretching), respectively (Amaral et al., 2005). In the spectrum of bared iron oxide (curve c), peaks appeared at 3485, 1635 and 590 cm1 were for O–H stretching, O–H bending and Fe-O stretching, respectively (Nanta et al., 2012). In the spectrum for MMT (curve d), peaks at 3398, 1638 and 1123–581 cm1 were for –OH stretching, –OH bending and Si–O bond, respectively (Su et al., 2008). The peaks exhibited in the spectrum for isoniazid (curve e) at 1650 cm1 and at 1558 cm1 were due to amide I (C¼O stretching) and amide II(NH bend), respectively. Besides these, multiple peaks were also appeared in the range of 1420–670 cm1 (Rastogi et al., 2007). The characteristics peaks of isoniazid, CMS, CS and MMT were appeared in drug-loaded nanoparticles (curve e) suggesting the affirmative coating of the iron oxide nanoparticles along with incorporation of MMT and drug. The intensity of O–H stretching peak in the drug-loaded nanoparticles decreased and shifted to

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Figure 2. FT-IR spectrum of (a) CMS, (b) CS, (c) Iron oxide, (d) MMT, (e) Isoniazid and (f) NP/M3/GA5.

lower wavenumber compared to iron oxide, CMS and CS indicating the interaction between CMS, CS and iron oxide nanoparticles (Figure 2). X-ray diffraction study The diffractogram of CMS and CS exhibited a broad peak at 2y ¼ 20 and 2y ¼ 22 , respectively (Zong et al., 2000; Zang et al., 2012). The diffractogram of iron oxide showed a high degree of crystallinity and presence of several diffraction peaks related to magnetite crystallographic phase at 2y ¼ 30 , 35 , 45 , 52 , 57 and 60 . These peaks were due to (220), (311), (400), (422), (511) and (440) plane respectively (Karaagac et al., 2010). Isoniazid showed multiple peaks at 2y ¼ 10–50 due to its crystallinity (Devi and Maji, 2010). The characteristics peaks of MMT exhibited at 2y ¼ 9.4 and 27.3 were assigned for (001) and (002) plane, respectively (Bahari et al., 2011). The characteristics peaks for MMT were found to be disappeared in XRD diffractogram of CMS-CS-coated iron oxide/MMT nanoparticles. Therefore, it could be said that either the full expansion of MMT gallery occurred which was not possible to detect by XRD or the MMT layers become delaminated and no crystal diffraction peak

appeared. The peak due to (311) plane at 2y ¼ 35 was found to appear in the diffractogram of CMS-CS-coated iron oxide/MMT nanoparticles. The suppression of the characteristics peaks of CMS and CS indicated their compatibility and good interaction in resulted PEC. The intensity of the crystalline peaks of iron oxide decreased due to introduction of CMS and CS polymers indicating that the crystallisation was effected by the polymer chains. Similarly, the disappearance of characteristics peaks of isoniazid in drug-loaded nanoparticles suggested the occurrence of molecular level dispersion of isoniazid in isoniazid-loaded PEC-coated iron oxide/MMT nanoparticles. Angadi et al. reported the molecular level dispersion of isoniazid in CS-hydroxyethyl cellulose blended microcapsules (Angadi et al., 2010) (Figure 3). Thermal property study of the nanoparticles The thermomagnetic study of the drug-loaded nanoparticles showed presence of magnetic phase in the nanoparticles. Figure 4(a) shows the variation of magnetic susceptibility with heating and cooling simultaneously. A complex alteration for the heating and cooling curves was found. They showed higher

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susceptibilities on heating curves. From these curves the Curie temperature for the coated nanoparticles was found to be at TC ¼ 553 , indicating that the formed nanoparticles were in magnetite phase (Levy et al., 2012). The attachment of PEC and MMT with the iron oxide nanoparticles was satisfied from the TGA analysis (Figure 4b). The initial weight loss occurred at around 50  C in both pure CMS and CS was due to the removal of residual solvent and water. For both neat polymers, major decomposition occurred in the temperature range 90–450  C with a weight loss of 68%

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(approximately). The loss was due to dehydration, depolymerisation and decomposition of the polymer chains. The curves showed a continuous dropping trend. After this stage, about 25% residue left in the polymers. Both pure iron oxide and MMT showed much more thermal stability than the polymers. They showed only 10–12% weight loss. PEC-coated iron oxide/MMT nanoparticles showed higher thermal stability than those of both of the pure polymers and lower than both MMT and iron oxide, suggesting the presence of polymer matrix on the surface of iron oxide nanoparticles. RW (%) value of these nanoparticles was also found in between to those of neat polymers, iron oxide and MMT.

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Morphology study of the nanoparticles SEM image (Figure 5a–b) shows that isoniazid-loaded nanoparticles have solid dense structure with spherical shape. The surface of the PEC-coated iron oxide nanoparticles appeared homogeneous indicating good compatibility between CM, CS and iron oxide. The surface of the nanoparticles with MMT appeared less smooth and agglomerated compared to that of MMT free nanoparticles. Similar results were reported by Dong and Feng (2005), while studying the morphology of paclitaxel-loaded PLGA/MMT nanoparticles. TEM image of isoniazid-loaded nanoparticles without MMT and with MMT are shown in Figure 5(c–d). The PEC-stabilised iron oxide nanoparticles showed excellent dispersion. The dark lines represented the intersection of MMT layers. Similar observation was reported by Choi et al. (2012). These results indicated that MMT was well incorporated in the nanoparticles. Swelling study

Figure 3. XRD spectrum of (a) CMS, (b) CS, (c) Iron oxide, (d) MMT, (e) Isoniazid and (f) NP/M3/GA5.

The results of swelling experiment of the nanoparticles at gastric pH (pH ¼ 1.2) and intestinal pH (pH ¼ 7.4) are shown in Figure 6(a–b). Swelling was more at higher pH compared to lower pH. At lower pH, the amino groups of CS were protonated, and then interacted with the carboxyl groups of CMS (Assaad et al., 2011). This would result into lower swelling. At higher pH (pH 7.4), the carboxyl groups of CMS were deprotonated and ionised, favouring hydration and thus better swelling. On the other hand, at higher pH, deprotonation of CS declined the extent of ionic interactions which leads into complex dissociation and hence resulting into increased degree of swelling. Again with increase in MMT concentration, the swelling degree was found to be decreased. The silicate layers of MMT hindered the penetration of water into the polymer matrix and thereby suppressed the swelling. Again, the nanoparticles containing glutaraldehyde

Figure 4. (A) Susceptibility curve of NP/M3/GA5 and (B) TGA curve of (a) CMS, (b) CS, (c) Iron oxide (d) MMT and (e) NP/M3/GA5.

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Figure 5. SEM image of (a) NP/M0/GA5 and (b) NP/M3/GA5, TEM image of (c) NP/M0/GA5 and (d) NP/M3/GA5.

swelled less than those of nanoparticles without glutaraldehyde. This might be ascribed to the formation of cross-linked structure by the glutaraldehyde. In vitro release studies Figure 6 (c–d) shows drug release profile from PEC-coated iron oxide nanoparticles in gastric pH (pH ¼ 1.2) and in intestinal pH (pH ¼ 7.4) environment. Figure 6 indicates the pH dependency of the drug release from the nanoparticles. Cumulative drug release (%) was found to be better in intestinal pH. The main factors governing the release of drug from nanoparticles are swelling nature of the polymers and solubility of the drug in the medium. It was already stated that alkaline pH favoured swelling of the PEC. Figure 6(e) shows the drug release in presence of magnetic field (0.2 T and 0.5 T) at pH 7.4. The presence of lower magnetic field (0.2 T) did not exhibit any significant improvement in release pattern compared to those without magnetic field. However, higher magnetic field enhanced the initial burst release by approximately 10%. In presence of magnetic field, drug release becomes faster due to magnetic constriction of the nanoparticles (Pereira et al., 2013). Isoniazid has high affinity for hydrolysis at pH 7.4 due to the presence of amine (basic) group. This might favour the release. Isoniazid was also reported to be a BCS (biopharmaceutical

classification system) class I category drug (BCS), i.e. it have higher solubility throughout the entire gastrointestinal pH conditions (pH 1–7.5). This solubility factor might also help in enhancing the higher release of isoniazid at pH 7.4. The release of drug from the nanoparticles was found to be decreased with the increase in MMT concentration. The silicate layers of MMT provided a tortoise path and hence decreased the drug release rate. Addition of crosslinker was also found to reduce the release of drug due to the formation of cross-linked structure. Therefore, the use of MMT and crosslinker was found to be beneficial in controlling the isoniazid release from the synthesised drug delivery system. Cytotoxicity test The result of MTT assay is shown in Figure 7. MTT assay was done for different concentration of the nanoparticles at 8 h. It was observed that CMS, CS and MMT alone did not show any significant cytotoxicity while isoniazid and iron oxide showed considerable amount of cytotoxicity. The polymer coating on nanoparticles slowed down the release of drug and hence decreased the interaction of drug with the cell wall. Therefore, PEC-coated iron oxide and isoniazid-loaded nanoparticles showed more cell viability than iron oxide and also isoniazid, respectively. The result of cell viability of nanoparticles having different

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Figure 6. Percentage swelling degree at (A) pH 7.4 and (B) pH 1.2 and Cumulative percentage drug release at (C) pH 7.4 and (D) pH 1.2 of (a) NP/M0/ GA5, (b) NP/M1/GA5, (c) NP/M2/GA5, (d) NP/M3/GA5, (e) NP/M5/GA5 and (f) NP/M3/GA0, (E) Drug release in presence of magnetic field (a) 0 T, (b) 0.2 T (c) 0.5 T.

concentration of MMT (Figure 7f–j) showed that cell viability increased with the increase of MMT concentration upto 3%, beyond that it decreased. This was due to the fact that addition of MMT increased the cell viability by providing hindrance to the drug release through its complex silicate layer. But at higher MMT concentration, cell viability decreased due to release of drug at faster rate caused by the collapsing of silicate layer of MMT. The incorporation of an optimum amount of MMT in iron oxide nanoparticles modified with PEC of CMS-CS exhibited excellent biocompatibility and hence it could be used as a convenient material for future biomedical application.

Mucoadhesive test Table 2 and Figure 8 show the results of in vitro wash off and ex vivo mucoadhesive test, respectively. The results presented are the mean value of five readings. The test was carried out in intestinal pH (pH ¼ 7.4) environment as the drug release was found better in pH ¼ 7.4. From in vitro study (Table 2), it was found that mucoadhesive property of CMS was greatly enhanced when it was combined with CS. Therefore, iron oxide nanoparticles coated with PEC of CMS-CS showed significantly good mucoadhesive properties. In ex vivo test, it was observed that the

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DOI: 10.3109/02652048.2014.940015

Figure 7. Cell viability study with variation of concentration of (a) CMS, (b) CS, (c) Iron oxide, (d) MMT, (e) Isoniazid, (f) NP/M0/GA5, (g) NP/M1/ GA5, (h) NP/M2/GA5, (i) NP/M3/GA5, (j) NP/M5/GA5.

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Table 2. Results of in vitro wash-off test to assess mucoadhesive properties of prepared nanoparticles. Sample code

0 5 min min

CMS CS PEC NP/M0/GA5 NP/M3/GA5

53 50 51 52 52

35 39 37 39 48

10 min

(±1)22 (±2)25 (±1)27 (±3)39 (±1)43

20 min

(±2)20 (±1)24 (±3)25 (±3)33 (±1)37

30 min

(±1)12 (±1)23 (±1)21 (±1)30 (±2)30

60 min

(±3) 8 (±2)16 (±1)15 (±2)27 (±5)25

90 min

(±1) 5 (±4)10 (±5)10 (±1)21 (±2)24

120 min

(±1) 2 (±2) 6 (±2) 9 (±2)18 (±1)20

(±2) (±4) (±1) (±1) (±1)

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Notes: Each value represents average of five readings, standard deviation in parenthesis.

observed by SEM study. TEM study also indicated that MMT was well dispersed in the nanoparticles. Higher pH and crosslinking favoured better swelling and drug release. Furthermore, presence of magnetic field facilitated the drug release. Cell viability increased with the increase in MMT concentration in the system. MMT incorporation was also found to be beneficial in getting better mucoadhesivity of the nanoparticles. It may be concluded that PEC of CMS-CS can be used as a proficient coating agent for the synthesis of iron oxide magnetic nanoparticles having optimum drug release properties, suitable cell viability and enhanced mucoadhesivity. These results suggest that this type of amalgamated magnetic nanoparticles can be used in future as targeting drug delivery agent.

Acknowledgements University Grant Commission (UGC) is acknowledged for financial support to C. Saikia in the form of institutional fellowship.

Declaration of interest This study was supported by University Grant Commission (UGC) in the form of institutional fellowship to C. Saikia. There are no conflicts of interest.

References

Figure 8. Ex vivo mucoadhesive test of (a) NP/M0/GA5, (b) NP/M1/ GA5, (c) NP/M2/GA5, (d) NP/M3/GA5 and (e) NP/M5/GA5.

detachment force increased with the increase of MMT concentration. The detachment force was also found to enhance with the increase in contact time. Addition of MMT enhanced the adhesivity of the nanoparticles with mucous. It might be due to development of London van der Waals forces and H-bonding between MMT and mucous cells. Due to the presence of large surface area, negative charge and hydrophilic properties, MMT possesses excellent bio-adhesivity (Galindo and Viseras, 2004). The influence of MMT on bioadhesion in PLGA/MMT nanocomposite was shown by Dong and Feng (2005). The enhanced mucoadhesivity of PEC-coated iron oxide nanoparticles indicated its suitability in drug delivery applications.

Conclusion Isoniazid was successfully incorporated in MMT containing PECcoated iron oxide nanoparticles. The incorporation of an optimum amount of MMT enhanced both drug loading and encapsulation efficiency. Crosslinker also enhanced the stability of the nanoparticles. The size of the particles was in nanometer range as judged by DLS and TEM study. The incorporation of MMT as well as the coating of iron oxide nanoparticles with PEC of CMSCS was revealed by FTIR. XRD study indicated the incorporation of MMT and molecular level dispersion of isoniazid in PECcoated iron oxide nanoparticles. Thermomagnetic study showed that the nanoparticles were in magnetite phase. TGA study showed an improvement in thermal stability of PEC-coated iron oxide nanoparticles. Nanoparticles without MMT were smooth and spherical compared to the nanoparticles containing MMT as

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