Synthesis and characterization of chitosan-coated magnetite nanoparticles and their application in curcumin drug delivery

In this work anti-cancer drug curcumin-loaded superparamagnetic iron oxide (Fe3O4) nanoparticles was modified by chitosan (CS). The magnetic iron oxide nanoparticles were synthesized by using reverse micro-emulsion (water-in-oil) method. The magnetic nanoparticles without loaded drug and drug-loaded magnetic nanoparticles were characterized by XRD, FTIR, TG-DTA, SEM, TEM, and VSM techniques. These nanoparticles have almost spherical shape and their diameter varies from 8 nm to 17 nm. Measurement of VSM at room temperature showed that iron oxide nanoparticles have superparamagnetic properties. In vitro drug loading and release behavior of curcumin drug-loaded CS-Fe3O4 nanoparticles were studied by using UV-spectrophotometer. In addition, the cytotoxicity of the modified nanoparticles has shown anticancer activity against A549 cell with IC50 value of 73.03 μg/ml. Therefore, the modified magnetic nanoparticles can be used as drug delivery carriers on target in the treatment of cancer cells.


Introduction
Chemotherapy is not specialized to a certain treatment, which is the most notable drawback of this therapy method. When drug goes into the body, the medicine does not concentrate mainly on the diseased cells. Hence, magnetic particles are recently used to carry the drugs to the required location in the body, typically cancer tissues. The method of using superparamagnetic iron oxide nanoparticles has received much attention from the research community with two main objectives: (i) to narrow the distribution of drugs in the body for reducing the side effects; (ii) to reduce the amount of drug consumption.
The biocompatible magnetic nanoparticles are encapsulated with drugs. They work as drug delivery carriers and control the drug release process. Therefore, the application of superparamagnetic iron oxide nanoparticles in diagnosis and therapeutics has gained many promising achievements such as cell separation [1], cell apoptosis [2], and enzyme immobilisation [3]. Generally, the drug-particle system creates a magnetic liquid and enters the body via the circulatory system. When the particles enter the blood vessel, a strong external magnetic field gradient is used to guide the particles to arrive at the targeted location in the body. When the drugparticle system is concentrated at the required location, the | Vietnam Academy of Science and Technology Advances in Natural Sciences: Nanoscience and Nanotechnology Adv. Nat. Sci.: Nanosci. Nanotechnol. 7 (2016)  Original content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. drug release process is taken place by enzyme activity or physiological property change of cancer cells causing the variation of pH, diffusion, and temperature. Drug carried by magnetic nanoparticles can easily be directed to a targeted location in the body by magnetic force [4,5]. Meanwhile, the polymer coated drug can delay the releasing speed. Therefore, the magnetic nanoparticles coated polymer system are considered to be an effective method to target the cancer cells.
The magnetic material is iron oxide covered by organic or inorganic molecules which form chemical bonds with the surface of Fe 3 O 4 nanoparticles. Iron oxide core particles are nanometer-size superparamagnetic particles. Modified magnetic nanoparticles have been studied by different researchers [6,7] using 3-aminopropyl triethoxy silane (APTES) coated by nano-carrier through adsorption or covalent bond creating active amino groups to carry anti-cancer drugs. Yao et al [8] prepared magnetic Fe 3 O 4 /SiO 2 -GO core/shell nanoparticles by means of covalent and used as adsorbent. Their results show that this material has a relatively high adsorption ability. In another study, Peng et al [9] reported a simple method to synthesize mesoporous nanoparticles of core-shell structure Fe 3 O 4 -@mZnO to use as drug delivery carriers.
Curcumin, an anti-cancer drug, is derived from phenol which is connected by two α, β-unsaturated carbonyl groups. The diketones form stable enols and are readily deprotonated to form enolates. The α, β-unsaturated carbonyl group is a good michael acceptor and undergoes nucleophilic addition. Therefore, they are biologically characterized as antioxidant [10], anti-inflammatory [11], antibacterial [12], and antitumor activity [13]. However, solubility of curcumin in water is low, unstable and the short half-life cycles of in vitro metabolism limited its clinical application. To increase the solubility in water and biological compatibility of curcumin, different carriers were tested to encapsulate drug in polymer micelles [14], solid-lipid particles [15], and nano-polymeric particles [16].
Among the available polymers, natural chitosan shows some promising results in drug delivery system. Chitosan (CS), the second most naturally abundant polymer after cellulose, is a natural bio-polymer derived from alkaline deacetylation of chitin. Due to its chemical structure and the nontoxic nature [17], CS has received much attention and used widely in many fields such as adsorbent [18] and anti-bacterial membrane [19]. In some recent applications, CS is often used in drug delivery system [20][21][22] and protein carriers [23].
Many different methods have been investigated to synthesize magnetic nanoparticles such as co-precipitation, solgel process, micro-emulsion, ultrasonic chemistry, hydrothermal, hydrolysis, thermolysis, and flow injection method. The formation of nanoparticles depends on the synthetic method. Micro-emulsion method has high capability to disperse Fe 3 O 4 nanoparticles in a solution and particle size can be well controlled by the surfactant.
In this study, superparamagnetic Fe 3 O 4 nanoparticles were synthesized by micro-emulsion method used as carriers.
The carrier was coated as chitosan to create a polymeric shell to form an active amino group on its surface to be able to combine with the anti-cancer curcumin (Cur) drug. The morphology, structure, and characteristic of iron oxide nanoparticles coated with CS solution were studied by x-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), thermogravimetric analysis (TGA), differential thermal analysis (DTA), and vibrating sample magnetometer (VSM). The application of chitosanloaded magnetic nanoparticles (CS-MNPs) as carrier of Cur was evaluated by loading and releasing profiles and in vitro cytotoxicty.

Synthesis of magnetic Fe 3 O 4 nanoparticles
The Fe 3 O 4 magnetic nanoparticles were prepared by water-inoil micro-emulsion method. The precursor solution (solution A) contains 2:1 mole ratio of iron salts (10 ml of 0.4 M FeCl 3 .6H 2 O and 10 ml of 0.2 M FeSO 4 .7H 2 O) dissolved in 45 ml mixture of tween-80/butan-1-ol/n-heptane. This resulted in the formation of a reverse micro-emulsion. Mixture B contains 45 ml of tween-80/butan-1-ol/n-heptane added to aqueous NH 3 (56 ml of 28% aqueous NH 3 ). These solutions were stirred with speed of 300 rmp/min for 30 min at room temperature. Mixture B was added to solution A and the combined mixture was stirred continuously with a rotational speed of 1000 rpm for 24 h at different temperatures were 30, 50, and 80°C. Formed magnetic nanoparticles were separated by magnetic force and washed several times by distilled water to eliminate the ammonia. Finally, the magnetic Fe 3 O 4 nanoparticles were obtained after drying in a vacuum oven, and recorded as Fe 3 O 4 -30, Fe 3 O 4 -50, and Fe 3 O 4 -80, respectively.

Synthesis of chitosan-coated magnetic Fe 3 O 4 nanoparticles
The surface of magnetic Fe 3 O 4 nanoparticles was coated with a solution of CS for the purpose of obtaining modified magnetic nanoparticles. In a typical experiment, 0.25 g of magnetic Fe 3 O 4 nanoparticles was dispersed in a surfactant containing CTAB (2 grams of CTAB dissolved in 400 ml of deionized water) (solution C). Then, 100 ml chitosan solution (0.02 gram CS powders dissolved in 100 ml of 1% (w/v) acetic acid solution) was slowly dropped into solution C. The mixture was continuously stirred with a rotational speed of 1000 rpm for 1 h at room temperature. Then, CS coated by magnetite nanoparticles was magnetically separated from solution by a magnet bar and thoroughly washed several times with ethanol and deionized water. Finally, the obtained nanoparticles were dried overnight at 60°C.

Characteristics of materials
Powder x-ray diffraction patterns were recorded on a D8-Advance Bruker with Cu-Kα radiation (λ=1.5406 nm). Morphological analysis was examined on a Hitachi S-4800 scanning electron microscope (SEM). The images of TEM were taken by a JEM-1010 (Jeol, Japan) operated at an accelerating voltage of 200 kV. Infrared data were examined on KBr pallets by using a Shimadzu IR Prestige-21 spectrometer (Japan). TGA and DTA were performed with a TG Setaraminstrument (France) in the air of argon (100-1000°C). The magnetization of the prepared nanoparticles was measured on a VSM (DMS-88) at room temperature.
UV-vis and UV-vis-diffuse reflectance spectra were collected on a DR-Jasco V630 or a UV-vis DRS-Jasco V670 spectrophotometer that was equipped with a diffuse reflectance attachment in which BaSO 4 was the reference.

In vitro curcumin drug loading and release experiments
Anti-cancer curcumin drug was loaded on magnetic Fe 3 O 4 nanoparticles coated by chitosan (CS). Loading of curcumin was performed by dispersing 0.3 gram of chitosan-coated magnetic Fe 3 O 4 nanoparticles (CS-Fe 3 O 4 ) nanoparticle in 0.1% curcumin solution (dissolved in ethanol) and stirred for 3 h to increase the uptake of curcumin. At each fixed period of time, the magnetic nanoparticles were separated from the mixture by using a magnet. The absorbance of the residual curcumin in the supernatant was measured at λ max =428 nm by UV-vis spectrophotometer to determine the drug concentration of curcumin. The curcumin loading was determined from the difference between the initial concentration of curcumin in solution and concentration of curcumin in the supernatant solution after the set time interval.
The release profile of the curcumin drug studied in phosphate buffered saline (PBS) at pH of 7.4. 0.3 g of Cur-CS-Fe 3 O 4 was dispersed in PBS at 37°C and shaking slightly at a rate of 150 cycles/min. At each time intervals, the concentration of curcumin release in the PBS was analyzed by UV-vis spectrophotometer.
2.6. In vitro cytotoxicity assay A549 cancer cell lines were cultured as monolayers in the culture medium of Dulbecco's modified eagle medium (DMEM) along with other components including 2 mM L-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 1.0 mM sodium pyruvate. 10% fetal bovine serum (FBS, Gibco) was also added. The cells were cultured and moved after 3-5 days with a ratio of 1:3 and cultured in CO 2 incubator at 37°C with 5% CO 2 . Monks's method [24] was implemented for vitro cytotoxicity. The test was conducted to determine the amount of total cell protein based on the optical density (OD) measured the protein components of the cell stained with the sulforhodamine B (SRB). 20 μl of the A549 cells diluted in 10% of dimethyl sulfoxide (DMSO) was put into the standard 96-well plates to obtain different concentrations of 100 μg ml −1 , 20 μg ml −1 , 4 μg ml −1 , and 0.8 μg ml −1 . Trypsinized cells were used to separate and count cells in the counting chamber to obtain a suitable experimental density. A suitable number of cells was added to the wells (in 180 ml medium) and so they can develop within 3-5 days. Other 96-well plates without the A549 cells containing 180 μl cancer cells were used to control day 0. After 1 h, the cells in the control plate of day 0 were immobilized by trichloracetic acid (TCA). After a period of development in the CO 2 incubator, the cells were immobilized to the bottom of the well by TCA and stained by SRB in 1 h at 37°C. After removing the SRB, the experimental wells were washed 3 times with acetic acid and dried by air at room temperature.
Finally, 10 mM unbuffered tris base was used to dissolve the SRB sticking on the protein molecules. The system was put on a shaker plate shaking gently for 10 min Then, elisa plate reader (bio-rad) was used to read the color content of the SRB through UV-vis at λ max =540 nm. The cell viability (%) was related to the control wells containing untreated cells with fresh cell culture medium. The cell viability was calculated by the following formula:  Broadness of the diffraction peaks was related to particle sizes. Scherrer's equation was used to calculate the average particle size D. In this equation θ is the angle of the peak, β is the full width at half maximum (FWHM) of the respective XRD peak, λ is the x-ray radiation wave-length in angstroms, and k is a constant.     ). This stage may be the evaporation of water or OH groups adsorbed on the surface of the iron oxide. In the second stage, the weight loss of 3.7% corresponds to the broad exothermic peak at 235°C (curves b in figures 7(A) and (B)). The cause of this occurrence is due to the oxidation products of Fe 3 O 4 that are easily oxidized to give γ-Fe 2 O 3 particles [29].
The weight loss of 12.7% corresponds to the strong exothermic peak at 433°C (curves c in figures 7(A) and (B)). This shows that the OH groups on the surface of the magnetic nanoparticles are covalently bonded to the NH 2 groups in chitosan molecules. Hence, it could infer that the weight loss was the release of hydroxyl ions and decomposition of chitosan on the Fe 3 O 4 nanoparticles except water thermodesorption. It was also confirmed that the Fe 3 O 4 nanoparticles were successfully coated by chitosan. In addition, the sharp   nanoparticle is due to the decrease in particles size. Along with that, the particle size increase is due to the polymer layer coated on the surface of the particles and this also leads to a decrease in the value of magnetic saturation.
The results of vibrating sample magnetometer (VSM) show that all particles have superparamagnetic properties at room temperature since the zero coercivity and the remanence are almost negligible in the absence of an external magnetic field. According to the study by Mohapatra et al [30], Yavuz et al [31], superparamagnetism exhibited in the nanopaticles is due to their size effect (<50 nm). The average particle diameter of prepared samples is smaller than the critical size of superparamatic Fe 3 O 4 at room temperature. The Fe 3 O 4 particles are considered to be at their superparamagnitism. On the other hand, it is considered that the superparamagnetic properties may also be due to the high crystallinity of the prepared spherical shape magnetic nanoparticles.  9(b)) shows the rapid adsorption of curcumin in the initial stages and the adsorption rate slowed down after 90 min Because the surface of the nanoparticles is incorporated by curcumin, the final saturation point was reached at 120 min. In addition, the adsorption time of 120 min did not significantly change the concentrations of curcumin because curcumin with the nanoparticles had reached the saturation point. From the results obtained above, it can be concluded that the maximum drug absorption time is short, about 120 min. Figure 10 depicts the in vitro drug release profile of curcumin from Cur-CS-Fe 3 O 4 nanoparticles at the pH of 7.4 and the release in phosphate buffer solution, at 37°C.
The UV-spectra of curcumin loaded CS-Fe 3 O 4 nanoparticles dissolved in buffer show an increase in absorbance with the increase in time. However, the release rate is relatively slow when the time was prolonged. The drug release rapidly occurs for the first 180 min. This may be due to the excess of curcumin drug molecules dispersed from matrix of the magnetic nanoparticles into buffer solutions, leading to the drug release at a faster rate [32]. The slow drug release was attributed to the presence of highly soluble phenolic acid groups in ionized drugs and the concentration reduction of chitosan on the surface [33].
About 30% of the un-released curcumin remining on the magnetic nanoparticles matrix for 2800 min may be due to inter-molecular hydrogen bonding of curcumin and also because chitosan has hindered the drug release from the particles [34,35]. This result is similar to that of the previous study by Ramanujan et al [34]. Drug loading and release profiles of PVA coated iron oxide nanoparticles showed that up to 45% of adsorbed drug was released in 80 h. Thus, the results show that Cur-loaded CS-coated iron oxide  nanoparticles are promising magnetic drug carriers to use in magnetically targeted drug delivery.

In vitro cytotoxicity assay
The cytotoxicity of free Cur and magnetic Cur-CS-Fe 3 O 4 nanoparticles evaluated against A549 cell lines with the concentrations ranging from 6.25 μg ml −1 to 100 μg ml −1 is presented in table 1. It was observed that cell inhibition was decreased at lower concentrations of nanoparticles. That is due to the lower concentrations of drug loaded into nanoparticles. The cancer cells treated with a lower concentration (10 μg ml −1 ) of the curcumin loaded magnetic Fe 3 O 4 nanoparticles show a low percentage of inhibitions (4.98%) whereas curcumin loaded magnetic nanoparticles with higher concentrations (100 μg ml −1 ) have a very high ability to inhibit cancer cells (78.55 μg ml −1 ). With this concentration, the free-curcumin inhibited cancer cells were 94.4%. Because of the slower release rates of curcumin from nanoparticles, it reduces the interaction of the drug with a cell wall.
From the obtained results, table Curve software was used to calculate the IC 50 values. IC 50 is the drug concentration at which the drug includes 50% inhibition of the growth of the cells. The results obtained from the two samples of freecurcumin and magnetic Cur-CS-Fe 3 O 4 nanoparticle shown the activity on cancer the A549 cell line with an IC 50 value of 73.03 and 11.37 μg ml −1 , respectively. It suggests that chitosan-coated magnetic nanoparticles have no cytotoxicity at the normal concentration and show a good biocompatibility.

Conclusions
Magnetic Fe 3 O 4 nanoparticles are synthesized by a reverse micro-emulsion of water-in-oil. The synthesized magnetic