Effect of crosslinker on drug delivery properties of curcumin loaded starch coated iron oxide nanoparticles

Accepted Manuscript Title: Effect of crosslinker on drug delivery properties of curcumin loaded starch coated iron oxide nanoparticles Author: Chinmayee Saikia Monoj K. Das Anand Ramteke Tarun K. Maji PII: DOI: Reference:

S0141-8130(16)31619-1 http://dx.doi.org/doi:10.1016/j.ijbiomac.2016.09.043 BIOMAC 6510

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

14-3-2016 12-9-2016 14-9-2016

Please cite this article as: Chinmayee Saikia, Monoj K.Das, Anand Ramteke, Tarun K.Maji, Effect of crosslinker on drug delivery properties of curcumin loaded starch coated iron oxide nanoparticles, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2016.09.043 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effect of crosslinker on drug delivery properties of curcumin loaded starch coated iron oxide nanoparticles Chinmayee Saikia1, Monoj K Das2, Anand Ramteke2, Tarun K. Maji1* 1,*

Department of Chemical Sciences, Tezpur University, Assam, 784028, India

2

Department of Molecular Biology and Biotechnology, Tezpur University, Assam, 784028,

India

*Corresponding author: Tel.: +91 3712 267007; Extn: 5053; Fax: +91 3712 267005. E-mail address: [email protected] (T. K. Maji).

Abstract Aminated starch coated iron oxide magnetic nanoparticles loaded with curcumin were synthesized via coprecipitation technique. The nanoparticles were crosslinked by using three different crosslinkers: glutaraldehyde, genipin and citric acid and the effect of crosslinking on different properties of the nanoparticles was evaluated. Characterisation of the nanoparticles was done with FTIR (Fourier Transform Infrared spectroscopy) and XRD (X-Ray Diffraction). Magnetic property study using VSM (Vibrating Sample Magnetometer) showed their superparamagnetic nature. Morphology of the nanoparticles was studied by SEM (Scanning Electron Microscopy) and TEM (Transmission Electron Microscopy). Zeta potential values showed that crosslinking imparted stability to the system. Crosslinking also enhanced drug loading and encapsulation efficiency of the system. Swelling and in vitro studies of the nanoparticles showed that the release of drug was dependent on time, crosslinker nature, crosslinker concentration and pH of the medium. The aminated starch coated nanoparticles also showed good mucoadhesive character. The cell viability assessment by MTT study revealed their compatibility with human lymphocytes cells and their considerable cell growth inhibiting properties with MCF7 and HepG2 cells. The nanoparticles showed good internalization in HepG2 cells along with considerable ROS formation. 1

Keywords: Aminated starch, iron oxide, curcumin, nanoparticle, drug delivery

1. Introduction Curcumin, an orange crystalline powder derived from the herb Curcuma longa (turmeric), a natural phytochemical with promising medicinal properties has been evaluated in different biomedical areas [1]. Curcumin exhibits immense therapeutic values including antioxidant, antiinflammatory, anti-microbial, anti-neoplastic and chemopreventive properties. Another advantage associated with curcumin is its minimal side effect as drug; it is safe even at higher doses [2]. Besides these advantages, its clinical applications are limited due to its poor bioavailability, poor solubility and ease of degradation at alkaline pH. Among the different approaches for minimization of these limitations, entrapment of curcumin within polymer matrix method has been widely accepted [3]. In this technique, the solubility and bioavailability of curcumin can be enhanced by entrapping it within hydrophilic biocompatible polymer and thereby controlling its release. Polymers with different functional groups offer many advantages in terms of better polymer- drug interactions leading to superior drug loading and encapsulation efficiency of the system, better mucous- polymer interaction, functionalization with targeting ligand leading to better targeted therapy etc.[4]. Some functional groups offer charge to the polymer backbone leading to an improvement in properties. Positively charged ones has been found to show greater cellular uptake, internalization and mucoadhesivity than both negative and neutral particles [5]. In cancer treatment, localization of the therapeutic material in cancer cells is of utmost interest. It reduces the chance of normal cells to be affected by the drug molecules. To achieve this challenge, considerable interest has been paid to drug delivery systems conjugated with a magnetic moiety. The main advantage of magnetic nanoparticles over nonmagnetic nanoparticles is their ability to localize the therapeutic agent within targeted cells or tissues [6]. The magnetic property of the nanoparticles helps them to convey a drug to specific tumor site under the influence of external magnetic field. The magnetic particles preferentially should be ‘superparamagnetic’ so that they are magnetic only under external magnetic field and then become inactive once the magnetic field is removed. The ‘superparamagnetism’ highly depends 2

on the size of the material; the particles should be of ultra-small size, less than 100 nm [7]. Therefore very much precaution should be made in order to maintain their size within such narrow diameter. Coating of the magnetic particles with polymer matrix is one of the most explored practices to get ultra-small nanoparticles. The polymer layer over the magnetic particle restricts their tendency of aggregation thereby limiting the size. The size can be furthermore limited by crosslinking the polymer matrix. Crosslinking can highly influence the encapsulation efficiency and swelling behavior of the matrix and thus significantly influence the release of the incorporated drug from the matrix. Different chemicals, both synthetic and natural origin have been explored to crosslink polymer chains. Glutaraldehyde, a synthetic crosslinker, has been studied as a successful crosslinker for polymer [8]. However its applications are narrow due to its toxicity. Genipin a natural crosslinker can successfully crosslink polymer containing amine groups and it is nontoxic [9]. The use of, citric acid, a natural crosslinker has also been reported [10]. In our study attempts have been made to compare the effects of these three crosslinking agents on release characteristics of curcumin from curcumin loaded aminated starch coated iron oxide magnetic nanoparticles. 2. Materials and methods 2.1. Materials Starch, ethylene diamine, Tween 80, and glutaraldehyde were obtained from Merck, India. Curcumin, montmorillonite K- 10, [3-(4, 5 dimethylthiazol-2-yl)- 2, 5- diphenyl tetrazolium bromide] (MTT) (M- 5655) were purchased from Sigma Aldrich, Germany. Epichlorohydrin was purchased from SRL, India. RPMI-1640, FBS (fatal bovine serum), DMEM and Penicillinstreptomycin antibiotics were purchased from HiMedia Laboratories, India. HepG2 cell lines and MCF7 cell lines were obtained from NCCS, Pune, India. Rest of the chemicals were of analytical grade and used as received. 2.2. Preparation of Curcumin loaded Aminated starch coated magnetic nanoparticles Starch was aminated by using the following procedure [11]. A solution of starch was made by dissolving 1 g of starch in a 50 mL of 0.1 M aqueous NaOH solution at 70 ºC in a 250 mL beaker. 0.1 mL of epichlorohydrin was added to the solution and kept for 7 h with constant 3

stirring under magnetic stirrer. Then the pH of solution was adjusted to 6-7. To this, 1 mL of ethylene diamine was added followed by addition of methanol. A white precipitate of aminated starch derivative was obtained. That precipitate was filtered and dried in vacuum oven. A solution of aminated starch derivative was prepared by dissolving 0.5 g in 25 mL of distilled water. In the meantime, solution of 0.35 g of FeCl3.6H2O and 0.15 g of FeCl2.4H2O were prepared in 25 mL of water and added to the above polymer solution at 60 ºC, under N2 gas atmosphere and kept for 1 h. Now, a solution of NaOH (0.1 M) was added dropwise to this solution under vigorous stirring at 60ºC for approximately 2 h until black precipitate occurred. The precipitate thus obtained was washed with water until the pH became less than 8.5. To load curcumin in the nanoparticles, 0.01 g of curcumin in 10 mL mixture of water and minimum amount of ethanol was added dropwise to 10 mL of an aqueous dispersion of nanoparticles. The mixture was then stirred overnight at 500 rpm on a magnetic stirrer in order to facilitate the penetration of curcumin into the polymer layers of the formulation. The drug loaded nanoparticles were then washed three times with water. Crosslinking of starch coated nanoparticles was done in three sets using varying amounts of three different cross linkers: glutaraldehyde, genipin and citric acid. In the first set, the obtained nanoparticles were then crosslinked by slow addition of (1%, 3% and 5%, v/w, w.r.t. polymer) glutaraldehyde/ or genipin/ or citric acid and the temperature was gradually raised to 45ºC. The reaction was continued for 1 h. The mixture was then cooled to room temperature. The product was filtered, washed and dried under vacuum. 2.3. Process yield calculation Process yield (%) was calculated by using the following formula: Pr ocess yield (%) 

Weight of nanoparticle 100 Weight of (drug  polymer )

2.4. Calculation of drug loading and encapsulation efficiency

4

At first a calibration curve of curcumin was drawn by taking absorbance values for known concentrations of curcumin (0.001 g- 0.01 g) at 420 nm by using UV- Visible spectrophotometer, model Hitachi, UV-2001, Japan. The nanoparticles were centrifused for 30 min and then the amount of free curcumin in the clear supernatant was determined by UV-Visible spectrophotometer at 420 nm. Then drug loading efficiency and drug encapsulation efficiency were determined by using the following formulae [12]: Loading efficiency (%) 

(Total amount of drug  Free amount of drug )  100 Weight of nanoparticle

Encapsulat ion efficiency (%) 

(Total amount of drug  Free amount of drug )  100 Total amount of drug

2.5. Characterisation of the nanoparticles Fourier transform infra-red (FTIR) spectroscopy study was conducted for coated magnetic nanoparticles and drug loaded magnetic nanoparticles with the help of Nicolet (model impact410) spectrophotometer scanned in the range of 4000- 400 cm-1. X-ray diffraction (XRD) study was also conducted for the iron oxide magnetic nanoparticles and the drug loaded nanoparticles with the help of Rigaku X-ray diffractometer (Miniflex, Japan) at a scanning rate of 1°/min using CuKα (λ=0.154 nm) radiation with an angle ranging from 2°- 70° of 2θ. Thermal properties of the nanoparticles were evaluated empolying a thermogravimetric analyser (TGA), model: TGA-50, Shimadzu, Singapore, at a heating rate of 5 ºC/min from 25 to 500 ºC under nitrogen atmosphere. A vibrating sample magnetometer (VSM) (model EverCool SQUID VSM DC magnetometer, Quantam Design, USA) was employed to study the magnetic properties of the synthesized nanoparticles at room temperature. To investigate the surface morphology of the nanoparticles, a Scanning electron microscope (SEM), (model: JEOL JSM-6390LV) at an accelerated voltage of 5-10 kV was used.

5

A transmission electron microscope (TEM), (model: JEOL JEM- 2100) operating at 100 kV was employed to determine the size and surface morphology of the nanoparticles. The Particle size distribution and zeta potential of drug loaded nanoparticles in aqueous medium were determined by a dynamic light scattering (DLS) analyzer (model DLS-Nano ZS, Zetasizer, Nanoseries, Malvern Instruments). 2.6. Swelling studies Swelling studies of the nanoparticles were determined in two different pH values (pH 5 and 7.4) by using the techniques available in literature [13]. Drug loaded nanoparticles were immersed in phosphate buffer solution 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: Swelling (%) 

( Final weight after swelling  Initial weight ) of the nanoparticless  100 Initial weight of nanoparticles

2.7. In vitro drug release studies In vitro drug release profiles from the nanoparticles were determined by dispersion technique at two different pH values (pH 5 and 7.4) [14]. The pH 5 was chosen for the study, as it is known that tumor tissues typically exists in acidic environment in the range of pH 4~ 5. Few papers cited the use of this pH for the drug release study in cancer cells [15-16]. Drug-loaded samples were immersed in phosphate buffer solution under continuous stirring. At scheduled time interval, 5 mL solution was withdrawn, filtered and the released drug was quantified spectrophotometrically at 420 nm 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. 2.8. Ex vivo mucoadhesive test The ex vivo mucoadhesive test was performed using Park and Robinson method [17]. The weight required to separate two-goat intestinal membrane from each other layered with test material between the surfaces, was determined by using a modified Robinson apparatus. In this method, keeping the mucosal side out, the intestinal membrane was secured on two glass vials using 6

nylon thread. The diameter of each mucosal membrane was 2 cm. The vials were then immersed in phosphate buffer at 37°C for 10 min for acclimation. A constant amount of nanoparticles was applied to the exposed tissue on the lower vial. The upper vial was adjusted so that the nanoparticles could adhere to the mucosal tissues of both the vials. Water was added at a constant rate to the pan on the other side of the modified balance until after a pre-determined period of time, the surfaces of the two membranes were separated. The weight of water represented the weight required for displacement. The adhesive force was then calculated using the following equation [18] Detachment stress (dyne.cm-2) = mg/A where “m” is the mass (g) required to detach the membrane, “g” is acceleration due to gravity taken as 980 cm/s2 and “A” is the area of tissue exposed, which is equal to πr2 (r is the radius of the exposed mucosal membrane). 2.9. Cell viability study The cell viability study was done on three types cells i.e. human lymphocytes, MCF7 (breast cancer cell) and HepG2 (liver cancer cell) cells. Human blood was anti-coagulated using Histopaque (1.077 g/mL) and lymphocytes were isolated from it. Aliquotes of 200 mL of the isolated cells were cultured in RPMI supplemented with 10 % heat inactivated fetal bovine serum (FBS). At first cells were maintained in RPMI for 4 h without FBS at 37 ºC in 5% CO2 in an incubator. Then they were treated with the nanoparticles and maintained in presence of FBS for 12 h [19]. Both MCF7 and HepG2 cell lines were grown in DMEM supplemented with 10% fetal bovine serum, 100 µg/mL penicillin-streptomycin antibiotics. Cells were maintained at 37°C and 5% CO2 in a humidified incubator and were sub cultured when they reached 80-90% confluence. Cell viability study was done using conventional MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay [20]. The cells were seeded in a 96 well plate at a density of 1×103 cells per well. Then the cells were incubated and allowed to adhere at 37 ºC for 48 h. After incubation, the medium was replaced with fresh medium and then treated with varying concentrations (50-150 µg/mL) of the samples. Control cells were treated with equivalent 7

volume of media. After 48 h, the media was discarded and replaced with 10 % MTT in phosphate buffer solution (PBS) followed by dissolving the formazan crystals in DMSO (100µL) and measuring the absorbance at 570 nm. The absorbance of control cells was separately set as 100 % viability and calculated as percentage control. 2.10. Cellular uptake studies Cellular uptake studies were done in HepG2 cell lines. The cells (1 x 105) were seeded in a six well plate in 2 mL medium containing 50 µM of the drug loaded nanoparticles. After 12 hours incubation time, cells were washed with PBS and stained with Prussian blue to detect the nanoparticle presence in the cancer cells. 2.11. Statistical analysis All the data were expressed as means ± SD. Results were statistically analysed by student’s t-test for significant difference between group mean using GraphPad software. The significant difference between the experimental and the control group was set as p<0.05. 3. Results and discussion 3.1. Effect of variation of crosslinker on different properties of nanoparticles Table 1 The effect of crosslinking on average size, stability, drug loading (%) and drug encapsulation efficiency (%) are shown in table 1. From the table, it was seen that the drug loading and encapsulation efficiency of the nanoparticles greatly enhanced due to crosslinking. Among the three crosslinking agents i.e. glutaraldehyde, genipin and citric acid, the nanoparticles crosslinked with genipin had shown maximum improvement in properties. It might be due to better crosslinking of the aminated starch with genipin. Both drug loading and encapsulation efficiency were found to increase with the increase in genipin concentration. The compact structure of the nanoparticles resulting from the crosslinking of polymer chains enabled them to load more amounts of drug molecules. The yield (%) was also found to enhance with crosslinking. It might be due to enhanced stability of the nanoparticles formed by the crosslinking of the polymer matrix [21]. Their stability could be judged from zeta potential values. Zeta potential for uncrosslinked nanoparticles was 10 mV which was then increased to 30 8

mV (with glutaraldehyde), 28 mV (with citric acid) and 35 mV (with genipin) after getting crosslinked. This value increased from 35 to 40 mV with the increase in genipin concentration (1-5%). Particle size was also affected due to crosslinking as seen from TEM study. Uncrosslinked nanoparticles showed an average particle size of 110 nm whereas crosslinked particles showed particle size in the range of 49- 63 nm. The highest particle size was observed for citric acid crosslinked nanoparticles. There was not much difference in the particle size between glutaraldehyde crosslinked and genipin crosslinked particles. The decrease in particle size was due to the formation of compact structure caused by the reaction of crosslinker with aminated starch. In the case of genipin, particle size was found to decrease upto 3% genipin concentration beyond that it increased further. It might be due to higher loading of drug molecule within the nanoparticles which resulted into increment of particle size. In the case of glutaraldehyde and genipin, a strong covalent bond might form between polymer and crosslinker. On the other hand, citric acid may produce weak bonds with polymer and as a result the nanoparticles would be less compact compared to those of both glutaraldehyde and genipin crosslinked particles and the particle size would be large. DLS study also displayed similar trend of variation in average particle size distributions. 3.2. FTIR studies Fig. 1 In the spectrum of curcumin (Fig. 1a), peaks appeared at 1600, 1510, 1446, 1265 cm-1 and at 1000 cm-1 were due to C=C of benzene ring, C=O and C-C vibrations, olefinic C-H bending vibration, aromatic C-O stretching vibration and C-O-C stretching vibration respectively [22]. The spectrum of bared iron oxide (Fig. 1b) showed characteristics peaks at 3269, 1635 and 575 cm-1 which could be assigned to O-H stretching, O-H bending and Fe-O stretching respectively [23]. In the spectrum of aminated starch (Fig. 1c), characteristics peaks appeared at 3396, 1625, 1485 and 874 cm-1 were due to -OH stretching and -NH bending, -NH stretching respectively. Presence of the characteristics peaks (with lower intensities) of curcumin, aminated starch, and iron oxide in drug loaded nanoparticles suggesting their incorporation. The intensity of O-H stretching peak in the genipin (3250 cm-1) (Fig. 1d), glutaraldehyde (3260 cm-1) (Fig. 1e) and 9

citric acid (3267 cm-1) (Fig. 1f) crosslinked drug loaded nanoparticles decreased and shifted to lower wavenumber compared to iron oxide (3269 cm-1) and aminated starch (3396 cm-1) indicating successful crosslinking between themselves. FTIR results also suggested that crosslinking was maximum and minimum for genipin and citric acid crosslinked nanoparticles respectively. 3.3. XRD studies Fig. 2 The diffractogram of curcumin showed multiple peaks at 2θ= 15-28° due to its crystallinity (Fig. 2a) [24]. Spectrum of iron oxide showed the presence of several diffraction peaks of magnetite crystallographic phase at 2θ= 30°, 35°, 43°, 50° and 59° (Fig. 2b) [25]. This spectrum matches well with a typical XRD pattern of magnetite nanoparticles. Aminated starch showed a broad peak around 2θ= 20° (Fig. 2c) indicating its amorphous nature. The disappearance of characteristics peaks of curcumin in drug loaded nanoparticles suggested the occurrence of molecular level dispersion of curcumin in curcumin loaded aminated starch coated iron oxide nanoparticles (Fig. 2d). 3.4. Magnetic properties Fig. 3 The magnetic properties of the nanoparticles were investigated by VSM. Fig. 3(I) shows magnetic moment vs temperature curve. Field dependent magnetization of the nanoparticles versus temperature was determined in the range of 2- 350 K with an applied magnetic field of 100 G by following standard zero field cooling (ZFC) and field cooling (FC) procedures. ZFC and FC analysis depict the cooling of the samples in absence of field and in presence of field respectively. Both nanoparticles with or without coating showed typical curve of superparamagnetic nanoparticles having blocking temperature of 238 K (curve a) and 148 K (curve b) respectively. The deviation in the curves above blocking temperature might be due to aggregation of the nanoparticles [26]. Fig. 3 (II) shows plots of magnetization verses applied magnetic field strengths of the iron oxide nanoparticles with and without polymer coating at room temperature. The sigmoidal shaped plot 10

without hysteresis loop indicated the superparamagnetic nature of the nanoparticles [27]. The saturation magnetization value had changed with the change in the amount of coating material. The saturation magnetization was found to be 80 and 42 emu/g for barred (curve a) and coated nanoparticles (curve b) respectively, indicating the presence of nonmagnetic polymer coating on the surface of magnetic iron oxide. These magnetic properties are very crucial for different biomedical applications. 3.5. Morphological studies Fig. 4 Fig. 4 (a-d) shows the SEM images of uncrosslinked and crosslinked nanoparticles. The surface of uncrosslinked iron oxide nanoparticles appeared agglomerated (Fig. 4a) while those of the crosslinked iron oxide nanoparticles had solid dense structure with regular surface (Fig. b-d). The surface of glutaraldehyde crosslinked nanoparticles showed floral like structure (Fig. 4b) while that of genipin crosslinked nanoparticles showed rod shaped structure (Fig. 4c). In the case of citric acid crosslinked nanoparticles no such regular shape was observed (Fig. 4d). TEM image for uncrosslinked nanoparticles (Fig. 4e) displayed the aggregation while TEM image for crosslinked nanoparticles showed dispersed particles with lower aggregation (Fig. 4f). 3.6. Swelling studies Fig. 5 Fig. 6 The swelling of the nanoparticles crosslinked with glutaraldehye, genipin and citric acid at pH 5 and pH 7.4 are shown in Fig. 5 and 6 respectively. Swelling increased with the increase in time. Swelling percentage decreased with the addition of crosslinker. Crosslinker formed compact structure and hence reduced the swelling (%). The variation in swelling percentage of crosslinked nanoparticles for the three different crosslinkers was as follows: citric acid> glutaraldehyde> genipin. The results indicated that crosslinking was higher with genipin compared to the other two crosslinkers. Genipin could react with the amine functional group to form covalent bonds, thereby forming a compact crosslinked structure.

The degree of

crosslinking with genipin was dependent on the pH of the reaction medium.28 In this study, 11

crosslinking was done under neutral condition thereby the linkages formed were short segment of crosslinking linkages. At neutral pH, ring opening of genipin took place that led to formation of heterocyclic amines caused by the nucleophilic attack of the amine groups of the polymer. Bifunctional glutaraldehyde could bind different reaction sites of aminated starch. It could form covalent imine bond with aminated starch.29 On the other hand under neutral condition, amine groups of aminated starch were supposed to possess positive charges which made it difficult to react with partially positive carboxyl groups of citric acid. Therefore the extent of crosslinking via citric acid would be less compared to those with other two crosslinkers. Due to better crosslinking with genipin, it was further chosen to study the swelling behavior of nanoparticles with varying genipin concentrations. With the increase in genipin concentration from 1- 5%, the swelling percentage was found to decrease in spite of the decrease in particle size. It might be due to the dominating effect of crosslinking which results into compact sized particles with network structure, over the size of the nanoparticles. It was also observed that swelling percentage was more at pH=5 than at pH= 7.4 irrespective of the type of crosslinker. In acidic medium, the amine groups of the polymer chains got protonated resulting in a polymer with high positive charge density. This charge formation facilitated the repulsion between polymer chains and hence improved the swelling behavior. 3.7. In vitro drug release studies Fig. 7 Fig. 8 The in vitro drug releases of the curcumin located crosslinked aminated starch coated iron oxide magnetic nanoparticles at pH 5 and pH 7.4 are shown in Fig. 7 and 8 respectively. From the study, it was observed that release percentage was depended on time, swelling characteristics and pH of the medium. Curcumin release increased with the increase in time and swelling percentage. At lower pH, release was more in comparison to that in higher pH. The order of release of drug with the three different crosslinker was as follows: citric acid> glutaraldehyde> genipin. Similarly, the release was found to decrease with the increase in the concentration of genipin. The decrease in drug release rate was due to lower swelling. With the increase in time, the degree of swelling enhanced the solvent access to the drug incorporated polymeric matrix, 12

and allowed greater release. The release rate exhibited by the nanoparticles crosslinked by different crosslinkers may be explained as stated earlier. In the case of uncrosslinked nanoparticles, an initial burst release of drug was observed and around 47% and 55% of drug was released within 8 h at pH 7.4 and pH 5 respectively. 3.8. Ex vivo mucoadhesion test Table 2 The ex vivo test for the genipin crosslinked nanoparticles was performed at pH 5 and pH 7.4 for three different time intervals i.e. 10 min, 20 min and 30 min (Table 2). The detachment force was higher at pH 5 than that at pH 7.4. This could be due to the presence of positively charged amine groups of the polymer matrix at lower pH which could strongly bind to the negatively charged mucous. The results indicated that the nanoparticles had good mucoadhesive properties. Again, mucoadhesivity was found to increase with the increase in genipin concentration. This might be due to formation of smaller sized particles. The higher the crosslinker amount, the lower was the particle size. Smaller particles provided higher surface area and hence increased the adhesion. 3.9. Cytotoxicity studies 3.9.1. In human lymphocytes Fig. 9 The results of MTT assay are shown in Fig. 9 (a) & (b). To check the cytotoxicity of the curcumin loaded magnetic nanoparticles in the concentration range from 50-150 µg/mL, human lymphocytes cells were incubated for 12 h. The nanoparticles as well as their ingredients i.e. aminated starch, iron oxide, curcumin and genipin showed good cell viabilities. The results indicated their good normal cell compatibility. Due to their natural origin, curcumin and genipin did not show toxicity to the cells and thus made them good candidates for chemotherapy. The MTT assay of the nanoparticles showed dose dependent cytotoxicity. 3.9.2. In MCF7 and HepG2 cell lines Fig. 10 The cytotoxicity of aminated starch, iron oxide, genipin, curcumin and curcumin loaded nanoparticles was assessed by MTT assay on HepG2 (Fig. 10 a & b) and MCF7 (Fig. 10 c & d) 13

carcinoma cell lines. The assay was done for the samples at three different concentrations i.e. 50, 100 and 150 µg/mL for 24 h. Aminated starch, iron oxide and curcumin did not show any toxic effect or marginal toxic effect whereas genipin and genipin crosslinked curcumin loaded nanoparticles showed some toxic effect on the cells. Genipin alone had shown some effectiveness in killing the cancer cells. Curcumin loaded genipin crosslinked nanoparticles showed dose dependent toxicity on both types of cancer cells. The toxicity increased with the increase in genipin concentration in the nanoparticles. As genipin alone had shown some amount of toxicity therefore it might influenced the nanoparticles to display the toxicity within the cells. The cytotoxicity provided by the nanoparticles could be drawn from their mucoadhesivity nature also. Since crosslinking enhanced the mucoadhesive properties of the nanoparticles, it facilitated better interaction of the curcumin loaded crosslinked nanoparticles with the mucin produced by the cancer cells. Mucoadhesivity of the nanoparticles enhanced their localization on the cancer cell surface and thus increased the accumulation of the released curcumin from the adhered nanoparticles to the inside of the cells. The better release of curcumin in acidic pH (pH 5) also facilitated the toxicity of the curcumin loaded nanoparticles. Fig. 11 Fig. 12 The percentages of live and dead cells were calculated by using haemocytometer with blue stain method. Effect of curcumin loaded nanoparticles on HepG2 and MCF7 cell morphology was noticed from images seen under inverted microscope (Fig 11 and 12 respectively). From the images it was observed that when cells were treated with polymer, iron oxide and curcumin, the cell morphology did not significantly change. Their cell structures were similar to those of the control. But the cell morphology significantly changed when it was treated with genipin. The cell morphology further changed on treatment with curcumin loaded magnetic nanoparticles. They inhibited the cell growth in both HepG2 and MCF7 cell lines. These results supported the findings by MTT assay. 3.10. Cellular uptake studies and ROS (reactive oxygen species) generation in HepG2 cell line Fig. 13 14

Fig. 14 The images of HepG2 cell lines treated with the nanoparticles are shown in Fig. 13. Satisfactory internalization of the nanoparticles was observed from the figures. Highest uptake was observed by NP/GE5 nanoparticles as revealed by visual analysis. NP/GE1 and NP/GE3 showed lesser amount of uptake. Generation of ROS plays an important role in oxidative damage of DNA, stimulating inflammation and apoptosis and all these roles form the basis for some anticancer drugs. The study of ROS generation by the prepared curcumin loaded nanoparticles exhibited a significant result as compared to the control (Fig.14). The generation of ROS increased from NP/GE1 to NP/GE5. The images further demonstrated the internalization of the nanoparticles in to the HepG2 cell (Fig.13). 4. Conclusion Development of safe and biocompatible drug delivery system along with controlled release properties having therapeutic properties is essential for cancer treatment. Curcumin loaded magnetic nanoparticles composed of iron oxide coated with crosslinked aminated starch was prepared. The impact of crosslinking on drug loading, particle size, swelling and drug releasing characteristics of the nanoparticles were studied using three different crosslinking agents: glutaraldehyde, genipin and citric acid. The best result was shown by genipin. At pH 5, the formulations showed good drug encapsulation efficiency, good controlled release properties with ~48% release upto 8 h. The formulations with varying concentrations of genipin showed that the drug release could be slowed down by increasing the genipin concentrations. Moreover, it exhibited good mucoadhesive properties and consequently improved cytotoxicity in MCF7 and HepG2 cancer cells. Genipin itself displayed cell growth inhibiting characteristics as revealed from MTT assay. More importantly its compatibility with human lymphocyte cells demonstrates that this formulation has the potential for biomedical applications. These nanoparticles also showd significant uptake in HepG2 cell line along with production of high amount of ROS. Conflict of interest There is no conflict of interest among the authors. 15

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[9] B. Manickam, R. Sreedharan, M. Elumalai, 'Genipin' - the natural water soluble cross-linking agent and its importance in the modified drug delivery systems: an overview, Curr. Drug Deliv. 11 (2014) 139-145. [10] J. Varshosaz, R. Alinagari, Effect of citric acid as crosslinking agent on Insulin loaded chitosan microsphere, Iranian Polym J. 14 (2005) 647- 656. [11] W.Y. Wang, Q.Y. Yue, X. Xu, B.Y. Gao, J. Zhang, Q. Li, J.T. Xu, Optimized conditions in preparation of giant reed quaternary amino anion exchanger for phosphate removal, Chem. Eng. J. 157 (2010) 1-167. [12] A. Tripathi, R. Gupta, S.A. Saraf, S, PLGA nanoparticles of anti tubercular drug: Drug loading and release studies of a water in-soluble drug, Int. J. Pharm. Tech. Res. 2 (2010) 21162123. [13] N. Devi, T.K. Maji, Microencapsulation of isoniazid in genipin-crosslinked gelatin-A-kcarrageenan polyelectrolyte complex, Drug Dev. Ind. Pharm. 36 (2010) 56-63. [14] R. Chahatray, D. Sahoo, D.P. Mohanty, P.L. Nayak, Guargum-Sodium Alginate Blended with Cloisite 30B for Controlled Release of Anticancer Drug Curcumin, World J. Nano Sci. Technol. 2 (2013) 26-32. [15] Z. Tong, W. et al., Tumor Tissue-Derived Formaldehyde and Acidic Microenvironment Synergistically Induce Bone Cancer Pain, PLoS ONE, 5 (2010), 1-15. [16] S. Y. Li, L. H. Liu, H. Z. Jia, W. X. Qiu, L. Rong, H. Cheng, X. Z. Zhang, A pH-responsive prodrug for real-time drug release monitoring and targeted cancer therapy, Chem. Commun. 2014, 50 (2014), 11852-11855. [17] K. Park, R. Robinson, Bioadhesive polymers as platforms for oral controlled drug delivery: method to study bioadhesion, Int. J. Pharm. 19 (1984) 107-127. [18] C.M. Lehr, J.A. Bowstra, J.J. Tukker, H.E. Junginger, Intestinal transit of bioadhesive microspheres in an in situ loop in the rat, J. Control. Release, 13 (1990) 51-62. [19] A. Hussain, V. Saikia, A. M. Ramteke, Free Radicals and Antioxidants 2 (2012) 8-11. [20] M. Das, D. Mishra, T. K. Maiti, A. Basak, P. Pramanik, Nanotechnology 19 (2008) 415101. 17

[21] F. Meng, Y. Zhong, R. Cheng, C. Deng, Z. Zhong, pH-Sensitive Polymeric Nanoparticles for Tumor-Targeting Doxorubicin Delivery: Concept and Recent Advances, Concept and Recent Advances Nanomedicine 9 (2014) 487-499. [22] A. Anitha, S. Maya , N. Deepa , K.P. Chennazhi , S.V. Nair & R. Jayakumar, , Curcumin loaded N,O- Carboxymethyl Chitosan nanoparticles for cancer drug delivery, J. Biomater. Sci. Polym. Ed. 23 (2012) 1381-1400. [23] P. Nanta, W. Sakolpap, K. Kasemwong, International Conference on Chemical and Environmental Sciences (ICCES’2012), Bangkok, 2012. [24] A. Anitha, S. Maya , N. Deepa , K.P. Chennazhi , S.V. Nair & R. Jayakumar, Curcumin loaded N,O- Carboxymethyl Chitosan nanoparticles for cancer drug delivery, J. Biomater. Sci. Polym. Ed. 23 (2012) 1381-1400. [25] O. Karaagac, H. Kockar, S. Beyaz, T. Tanrisever, A Simple Way to Synthesize Superparamagnetic Iron Oxide Nanoparticles in Air Atmosphere: Iron Ion Concentration Effect, IEEE Trans. Magn. 46 (2010) 3978-3983. [26] P. Guardia, B. Batlle-Brugal, A. G. Roca, O. Iglesias, M. P. Morales, C. J. Serna, A. Labarta, X. Batlle, Surfactant effects in magnetite nanoparticles of controlled size, J. Magn. Magn. Mater. 316 (2007) E756–E759. [27] N.T. Huong, L.T.K. Giang, N.T. Binh, L.Q. Minh, Surface modification of iron oxide nanoparticles and their conjuntion with water soluble polymers for biomedical application, Journal of Physics: Conference Series, 187 (2009) 1-5. [28] F.L. Mi, S.S. Shyu, C.K. Peng, Characterization of Ring-Opening Polymerization of Genipin and pH-Dependent Cross-Linking Reactions Between Chitosan and Genipin. J. Polym. Sci. Pol. Chem. 43(2005) 1985-2000. [29] I. Migneault, C. Dartiguenave, M. J. Bertrand, K. C. Waldron, Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking, BioTechniques, 37 (2004) 790-802.

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Caption of figures

Fig. 1 (a) Curcumin, (b) iron oxide, (c) Aminated starch, (d) Genipin crosslinked nps, (e) Glutaraldehyde crosslinked nps and (f) Citric acid crosslinked nps

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Fig. 2 (a) Curcumin, (b) Iron oxide, (c) Aminated starch, (d) Drug loaded crosslinked nps.

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Fig. 3 (I) ZFC- FC magnetization curve for (a) Iron oxide, (B) Coated iron oxide and (II) Magnetic hysteresis loop for (a) Iron oxide, (b) Coated iron oxide

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Fig. 4 SEM image of (a) Uncrosslinked nps, (b) Gluteraldehyde crosslinked, (c) Genipin crosslinked, (d) Citric acid crosslinked nps, and TEM image of (e) Uncrosslinked nps, (f) Crosslinked nps

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Fig. 5 Swelling of NP0, NP/CA, NP/GA, NP/GE1, NP/GE3, NP/GE5 (%) at pH 5

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Fig. 6 Swelling (%) of NP0, NP/CA, NP/GA, NP/GE1, NP/GE3, NP/GE5 at pH 7.4

24

Fig. 7 Cumulative release (%) of NP0, NP/CA, NP/GA, NP/GE1, NP/GE3, NP/GE5 at pH 5

25

Fig. 8 Cumulative release (%) of NP0, NP/CA, NP/GA, NP/GE1, NP/GE3, NP/GE5 at pH 7.4

26

Fig. 9 MTT assay in lymphocytes with 25, 50 and 100 µg/mL of different samples

27

Fig. 10 MTT assay in (a-b) HepG2 cell line and in (c-d) MCF7 cell line with 50, 100 and 150 µg/mL of samples

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Fig. 11 (a) Cell counting study on HepG2 cells and morphology of HepG2 cells after treatment with (b) control, (c) polymer, (d) iron oxide, (e) genipin, (f) curcumin, (g) NP/Ge1 & (h)NP/Ge5

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Fig. 12 (a) Cell counting study on MCF7 cells and morphology of MCF7 cells after treatment with (b) control, (c) polymer, (d) iron oxide, (e) genipin, (f) curcumin, (g) NP/Ge1 & (h)NP/Ge5

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Fig. 13 Cellular uptake in HepG2 cell line treated with (a) NP/GE1, (b) NP/GE3, (c) NP/GE5 and ROS study with (d) NP/GE1, (e) NP/GE3, (f) NP/GE5

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Fig. 14 ROS estimation on HepG2 cell line with NP/GE1, NP/GE3 & NP/GE5

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Table 1. Effect of variation of polymer concentration on different properties of the nanoparticles Sample

Cross

code**

linker (%)

Yield (%)

Loading

Encapsulation

Average diameterc

Zeta potentiald

efficiencya

efficiencyb

(nm)

(mV)

(%)

(%) TEM

DLS

NP/0

0

68 (±0.3)

58.5(±0.13)

50.2(±0.3)

110 (±4)

174 (±25)

0.10 (±0.01)

NP/CA

1

84 (±0.06)

61.2 (±0.2)

76.5 (±0.4)

63 (±5)

105 (±7)

28.12 (±0.02)

NP/GA

1

91 (±0.02)

64.8 (±0.3)

79.6 (±0.2)

50 (±2)

85 (±10)

30.10 (±0.04)

NP/GE1

1

90 (±0.2)

66.6 (±0.1)

80.6 (±0.6)

49 (±2)

83 (±6)

35.23 (±0.03)

NP/GE3

3

91(±0.4)

71.3(±0.03)

82.8(±0.03)

32(±3)

74(±10)

38.08(±0.04)

NP/GE5

5

94(±0.4)

74.0(±0.03)

84.1(±0.03)

37(±6)

79(±12)

41.00(±0.01)

Fe(II):Fe(III)=1:2, AS= 0.5 g, Curcumin= 0.01 g * a,

b, c, d: The value represents average of five readings, standard deviation in parenthesis

** in sample codes, NP denotes nanoparticle, CA denotes citric acid, GA denotes glutaraldehyde, GE denotes genipin

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Table2: Result of ex-vivo mucoadhesive test for NP/Ge1, NP/Ge3 & NP/Ge5 at pH 5 & pH 7.4' pH

Sample code

Time

Mass required

Detachment force

10 min

16.0

5012

20 min

16.7

5237

30 min

18.0

5617

10 min

17.8

5564

20 min

18.3

5711

30 min

18.8

5867

10 min

19.8

6179

20 min

20.1

6273

30 min

21.2

6616

10 min

9.9

3109

20 min

10.1

3158

30 min

10.3

3243

10 min

10.6

3339

20 min

10.7

3362

30 min

10.9

3411

10 min

10.10

3152

20 min

11.64

3634

30 min

11.67

3645

interval NP/Ge1

5

NP/Ge3

NP/Ge5

NP/Ge1

7.4

NP/Ge3

NP/Ge5

34