Fabrication of docetaxel surfaced Fe3O4 magnetite nanoparticles and their cytotoxicity on 4 T1 breast cancer cells
© Yazdi et al.; licensee BioMed Central Ltd. 2012
Received: 4 May 2012
Accepted: 17 May 2012
Published: 30 August 2012
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.
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.
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).
Today, side effects of anti cancer drugs are still considered as a major problem in chemotherapy of cancer diseases (). In the recent years, new drug delivery systems have been developed to reduce the side effects of these drugs ([2–4]). These systems mainly include nanotubes (), liposomes (), dendrimers () and nanoparticles (). The potential biomedical applications of Fe3O4 magnetite nanoparticles (MNPs) are well discussed in the literature (). Due to their magnetic properties, these nanomaretials have recieved particular attention as possible drug carriers (). The conjugation of chemical or natural anticancer drugs to Fe3O4 magnetite nanoparticles (MNPs) can lead to increse the uptake of anticancer agents by targeted tumor and improve their therapeutic index (). For example, a significant difference in antiproliferation effect of a natural product, umbelliprenin, and its Fe3O4 MNPs-conjugated form was observed in vitro model ().
Materials and methods
Synthesis of Fe3O4 magnetic nanoparticles and their characterization
A previously reported method was used for synthesis of Fe3O4 MNPs (). Briefly, deionized water (200 ml) was deoxygenated by bubbling nitrogen gas for 1.5 h, then 50 ml of NH4OH (1 M) was added and the mixture was stirred with mechanical agitation at 1000 rpm. Then, amounts of 6.76 g FeCl3.6H2O (2.5 mmol) and 4.97 g FeCl2.4H2O (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 Fe3O4 MNPs were separated from the solution. Freshly prepared Fe3O4 MNPs were characterized by transmission electron microscopy (model EM 208 Philips) and energy-dispersive spectroscopy (EDS).
Preparation of docetaxel-coated Fe3O4 nanoparticles and their surface characterization
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% CO2 at 37°C for one week and then used for cell proliferation assay.
Cell proliferation assay
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 Fe3O4 MNPs
Preparation and surface characterization of docetaxel coated Fe3O4 MNPs
The FTIR spectroscopy data of Fe 3 O 4 MNPs, docetaxel and docetaxel coated Fe 3 O 4 MNPs
Main peaks wave numbers (cm-1)
docetaxel-coated Fe3O4 MNPs
2922, 2927, 1736, 1246, 1161, 1065
3487, 3377, 2937, 1735, 1712, 1246, 1169, 1072
Literature review shows that there are numerous reports on cytotoxicity of docetaxel in combination of different carrier ([15, 16]). However, the efficiency of this compound in conjugation with Fe3O4 MNPs has not been yet investigated. Here, the antiproliferative effect of docetaxel enhanced in tumor cells when it was coated on the surface of Fe3O4 MNPs has been reported. As mentioned earlier, Fe3O4 MNPs, docetaxel and docetaxel-coated Fe3O4 MNPs, all showed antiproliferative effects on 4 T1 breast cancer cells, increasing in the order: docetaxel < Fe3O4 MNPs < docetaxel-coated Fe3O4 MNPs. It is noticeable that based on current study, docetaxel has considerable antiproliferative effect on 4 T1 breast cancer cells in concentrations of not below 10 μg/ml. However, obtained results demonstrated that when coated on Fe3O4 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 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. Therefore, it is deducible from Figure 5, that the concentration of 80 μg/ml docetaxel caused 79% cell death. However, when combined with Fe3O4 MNPs, the average concentration of 0.6 μg/ml docetaxel (which is equivalent to the concentration of about 20 μg/ml docetaxel- Fe3O4 MNPs) caused more than 90% cell death. Thus, it is concluded that by coating docetaxel on the surface of Fe3O4 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, Fe3O4 MNPs may be considered as efficient carriers for these insoluble compounds.
In this paper, the antiproliferative effect of docetaxel (IC50 =40 μg/ml) has been reported and the results revealed that the combination of Fe3O4 MNPs and docetaxel is significantly more cytotoxic (IC50 =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 Fe3O4 MNPs in other cell lines. Moreover, studies on cytotoxic effects of docetaxel-coated Fe3O4 MNPs in animal models will be of great value.
MHY: Collaboration in proliferation assays. ZNN: Collaboration in fabrication of nanoparticles. MRK: Commented on cytotoxic assays. MA: Commented on surface chemistry analysis of the nanoparticles. ARS: Design of the project. All authors read and approved the final manuscript.
This study was supported by Deputy of Research, Tehran University of Medical Sciences, Tehran, Iran.
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