Fabrication of docetaxel surfaced Fe3O4 magnetite nanoparticles and their cytotoxicity on 4 T1 breast cancer cells

Background In the recent years, there is an increasing attention to the using of Fe3O4 magnetite nanoparticles (MNPs) as drug delivery systems. Application of this nanoparticles could profit advantages of nanomedicine to enhance biological activity of pharmaceutical ingredients. Methods Fe3O4 MNPs were synthesised by a chemical method and characterized by transmission electron microscopy and energy-dispersive spectroscopy techniques. In the next step, docetaxel-coated Fe3O4 MNPs were prepared, using percipitation method. The surface chemistry of docetaxel-coated Fe3O4 MNPs as well as their thermal decomposition characteristics were examined using fourier transform infrared spectroscopy and thermogravimetric analyzer equipment, respectively. The cytotoxicity assay was conducted on 4 T1 breast cancer carsinoma by MTT assay to evaluate the possible in vitro antiproliferative effects of docetaxel-coated Fe3O4 MNPs. Results During precipitation process, docetaxel molecules were precipitated on the surface of Fe3O4 MNPs by the ratio of 3:100 w/w which indicates that each milligram of coated Fe3O4 MNPs averagely contained 30 μg pure docetaxel compound. Docetaxel showed aniproliferative effects against mentioned cell line. The higestest concentartion of docetaxel (80 μg/ml) caused about 80% cell death. However, the results demostarted that much lower amounts of docetaxel will be needed in combination of Fe3O4 MNPs to produce the potent antiproliferative effect compared to docetaxel alone. Dose response cytotoxicity assay of docetaxel-coated Fe3O4 MNPs against 4 T1 breast cancer cells showed that lower amount of docetaxel (0.6 μg/ml) can exhibit higher cytotoxic effect against this cancer cell line (90% cell death).


Introduction
Today, side effects of anti cancer drugs are still considered as a major problem in chemotherapy of cancer diseases (1). In the recent years, new drug delivery systems have been developed to reduce the side effects of these drugs (2)(3)(4). These systems mainly include nanotubes (5), liposomes (6), dendrimers (7) and nanoparticles (8). The potential biomedical applications of Fe 3 O 4 magnetite nanoparticles (MNPs) are well discussed in the literature (9). Due to their magnetic properties, these nanomaretials have recieved particular attention as possible drug carriers (10). The conjugation of chemical or natural anticancer drugs to Fe 3 O 4 magnetite nanoparticles (MNPs) can lead to increse the uptake of anticancer agents by targeted tumor and improve their therapeutic index (11). For example, a significant difference in antiproliferation effect of a natural product, umbelliprenin, and its Fe 3 O 4 MNPs-conjugated form was observed in vitro model (12). Docetaxel ( Figure 1) is an insoluble semi-synthetic analogue of Taxol. Docetaxel leads to a significant decrease in free tubulin, required for microtubule formation, and results in inhibition of mitotic cell division between metaphase and anaphase, preventing further cancer cell progeny (13)

Synthesis of Fe 3 O 4 magnetic nanoparticles and their characterization
A previously reported method was used for synthesis of Fe 3 O 4 MNPs (14). Briefly, deionized water (200 ml) was deoxygenated by bubbling nitrogen gas for 1.5 h, then 50 ml of NH 4 OH (1 M) was added and the mixture was stirred with mechanical agitation at 1000 rpm. Then, amounts of 6.76 g FeCl 3 .6H 2 O (2.5 mmol) and 4.97 g FeCl 2 .4H 2 O (2.5 mmol) were separately dissolved in 50 ml distilled water to make 0.5 M solutions. Subsequently, amounts of 10 ml ferrous chloride and 20 ml ferric chloride solutions were added to the ammonium hydroxide solution and the reaction mixture was stirred for 2.5 min at 1000 rpm. At this stage, a black precipitate formed which was washed four times with deionized water. Finally, using a magnet, the prepared Fe 3 O 4 MNPs were separated from the solution. Freshly prepared Fe 3 O 4 MNPs were characterized by transmission electron microscopy (model EM 208 Philips) and energy-dispersive spectroscopy (EDS).

Preparation of docetaxel-coated Fe 3 O 4 nanoparticles and their surface characterization
In order to coat docetaxel on the surface of Fe 3 O 4 MNPs, a simple precipitation method ( Figure 2) was used as follows: A 2 mg/ml dichloromethane solution of docetaxel (Cipla, Mumbai, India) was prepared and reserved in a sealed container. Fe 3 O 4 MNPs (100 mg) were dispersed in 5 ml distilled water. Thereafter, amount of 5 ml dichloromethane solution of docetaxel was added drop-by-drop to the suspension of Fe 3 O 4 MNPs over a 15 min period under continuously stirring (300 rpm) condition, and using a laboratory magnetic stirrer. The resulting mixture was maintained in a static regime at 45°C for 24 hours. Docetaxel-coated Fe 3 O 4 MNPs was separated from the solution by a magnet. Finally, the capped nanoparticles were washed three times with distilled water. A stock suspension of Fe 3 O 4 MNPs and docetaxel-coated Fe 3 O 4 MNPs were prepared (2 mg/ml) and used for antiproliferative bioassay. The surface chemistry of the docetaxel-coated Fe 3 O 4 MNPs was studied using Fourier transform infrared spectroscopy (Nicolet Magna 550). Also thermal decomposition characteristics of docetaxel-coated Fe 3 O 4 MNPs were determined using Thermogravimetric Analyzer TGA equipment as well.

Cell culture
The 4 T1 breast cancer cell line was obtained from the National Cell Bank of Iran (NCBI), Pasteur Institute of Iran, Tehran (Iran). The cells were cultured in a RPMI-1640 medium with 10% fetal calf serum, 100 u/ml penicillin and 100 μg/ml of streptomycin in 5% CO 2 at 37°C for one week and then used for cell proliferation assay.

Cell proliferation assay
Thiazolyl blue tetrazolium bromide assay (MTT) was performed to evaluate cell proliferation where 2 × 10 4 cells were seeded in 96 well plates and incubated with 0, 10, 20, 40 and 80 μg/ml of docetaxel-Fe 3 O 4 MNPs, bared Fe 3 O 4 MNPs or docetaxel alone for 24 hours. Then, 15 μl MTT (0.5 mg/ml) was added to each well, and the plates were incubated for 2 hrs. Then, the medium was replaced with 200 μl of DMSO and the absorbance was recorded at 570 nm (12). Each experiment was repeated three times and MTT assay was performed in 3 replicates for each experiments. The percentage of inhibition rate (IR) was measured using the following equation: Where ODexp and ODcon were optical densities of treated and untreated cells, respectively. Finally, The percentage-inhibition curve was obtained by plotting "the percentage of inhibition" (i.e.[absorbance of test wells/absorbance of control wells]/100) against the concentration of cytotoxic compounds, compared to control (i.e. untreated) cells.

Preparation and characterization of Fe 3 O 4 MNPs
As mentioned earlier, Fe 3 O 4 MNPs were chemically synthesized and their shape as well as size properties were confirmed by transmission electron microscopy. Upper illustration in Figure 3 demonstrates a representative TEM image of synthesized Fe 3 O 4 MNPs. TEM image shows that the particles range in size below than 20 nm and possess an average size of 10 nm. The lower

Antiproliferative effects
Antiproliferative effects of docetaxel-coated Fe 3 O 4 MNPs, docetaxel alone, and Fe 3 O 4 MNPs alone were in vitro evaluated at different concentrations on 4 T1 breast cancer cells. Cytotoxicity analysis of samples suggested a direct dose-response relationship, in which cell proliferation decreased in higher concentrations ( Figure 5). Samples were found to have different cytotoxicities on 4 T1 breast cancer cells, with docetaxel-coated nanoparticles being the most potent. The antiproliferative effect of test compounds increased in the following order: docetaxel < Fe 3 O 4 MNPs < docetaxel-coated Fe 3 O 4 MNPs. Moreover, the necessary concentrations to cause 50% cell death (IC 50 ) were more than 30 and 40 (μg/ml) in cells treated with Fe 3 O 4 MNPs alone and docetaxel, respectively. Docetaxel-coated Fe 3 O 4 MNPs could cause the same effect at 10 μg/ml concentration. In other words, the combination of docetaxel and Fe 3 O 4 MNPs exhibited a much more potent antiproliferative activity than docetaxel and Fe 3 O 4 MNPs alone. As clear in Figure 5, docetaxel at concentrations of 20 and 30 (μg/ml), moderately reduced the proportion of viable cells to 70% and 50%, respectively. The cytotoxicity curve for Fe 3 O 4 MNPs alone followed almost similar pattern, though sharper. The percentage of viable cell number sharply decreased from 87% at the concentration of 10 μg/ml to 35% at 40 μg/ml concentration. In contrast, however, only the low concentration of 10 μg/ml docetaxel-coated Fe 3 O 4 MNPs (equivalent to the concentration of approximately 0.3 μg/ml pure docetaxel), drastically reduced the proportion of viable cells by more than 70%. Also a considerable cytotoxicity (about 90%) was observed for 20 μg/ml docetaxel-coated Fe 3 O 4 MNPs against 4 T1 breast cancer cells. In higher concentrations (>20 μg/ml), this proportion declined steadily, until it reached the remarkably-low amount of about 7% at 80 μg/ml concentration.

Discussion
Literature review shows that there are numerous reports on cytotoxicity of docetaxel in combination of different carrier (15,16  antiproliferative effect on 4 T1 breast cancer cells in concentrations of not below 10 μg/ml. However, obtained results demonstrated that when coated on Fe 3 O 4 MNPs surface, docetaxel have antiproliferative effect, even at the low concentration of 0.3 μg/ml. This literally means that much lower amounts of docetaxel will be needed to produce the same antiproliferative effect as docetaxel alone. As described in above, during precipitation process, docetaxel molecules were precipitated on the surface of Fe 3 O 4 MNPs by the ratio of 3/100 w/w which indicates that each milligram of coated Fe 3 O 4 MNPs averagely contained 30 μg pure docetaxel compound. Therefore, it is deducible from Figure 5, that the concentration of 80 μg/ml docetaxel caused 79% cell death. However, when combined with Fe 3 O 4 MNPs, the average concentration of 0.6 μg/ml docetaxel (which is equivalent to the concentration of about 20 μg/ml docetaxel-Fe 3 O 4 MNPs) caused more than 90% cell death. Thus, it is concluded that by coating docetaxel on the surface of Fe 3 O 4 MNPs, the required amount of docetaxel for showing a potent antiproliferative effects against 4 T1 breast cancer cells could be considerably lowered (more than 100 fold). As mentioned before, many pharmaceutical compounds have low solubility in biological fluids, which reduces their efficiency in in vivo models. Therefore, it is hypothesized that, Fe 3 O 4 MNPs may be considered as efficient carriers for these insoluble compounds.

Conclusion
In this paper, the antiproliferative effect of docetaxel (IC 50 =40 μg/ml) has been reported and the results revealed that the combination of Fe 3 O 4 MNPs and docetaxel is significantly more cytotoxic (IC 50 =10 μg/ml) than docetaxel alone. In conclusion, this result is of particular value since it demonstrates that by using metallic nanoparticles in combination therapy, lower amounts of these materials might be required, resulting in reduction of the potential hazards of docetaxel usage. Further investigations are needed to determine the antiproliferative effect of docetaxel-coated Fe 3 O 4 MNPs in other cell lines. Moreover, studies on cytotoxic effects of docetaxel-coated Fe 3 O 4 MNPs in animal models will be of great value.