Synthesis of biocompatible iron oxide nanoparticles as a drug delivery vehicle

Introduction

Iron-based magnetic nanoparticles (NPs) such as magnetite (Fe₃O₄) have been studied in detail due to their unique properties, such as stability over time, biocompatibility, and sensitivity to magnetic field.^1–3 They can potentially be used as magnetic targeted drug delivery carriers and magnetic resonance imaging contrast agents due to their high saturation magnetization, low toxicity, and biocompatibility.⁴ The magnetic properties of Fe₃O₄ NPs are attributed to their size and size distribution, which, in turn, is dependent on the route of synthesis.

Therefore, in this study, an attempt was made to synthesize Fe₃O₄ NPs using a safe-by-design approach by the coprecipitation method. Polyethylene glycol (PEG) was used as surfactant to control the particle size and narrow size distribution. The biocompatibility of Fe₃O₄ NPs was evaluated by cytotoxicity assays and cell cycle analysis in the human breast adenocarcinoma cell line (MCF-7).

Materials and methods

Materials

Ferric chloride hexahydrate and ferrous sulfate were purchased from SD-Fine-Chem. Ltd, Mumbai, India. Ployetheleneglycol (PEG-6000), dimethylesulphoxide, sodium hydroxide (NaOH), minimum essential medium eagle, phosphate-buffered saline, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and antibiotic-antimycotic solution were purchased from HiMedia Laboratories Pvt. Ltd., (Mumbai, India). The MCF-7 cell line was purchased from the National Centre for Cell Sciences, Pune, India.

Fe₃O₄ NP synthesis

The preparation of Fe₃O₄ NPs was performed by a chemical coprecipitation method of Fe²⁺ and Fe³⁺ ions (1:2 molar ratios) by the addition of NaOH.⁵ A total volume of 15 mL of 0.25 M Fe²⁺ and 0.5 M Fe³⁺ solutions were prepared in deionized water. PEG was then added and the temperature slowly risen up to 80°C. During the initial 2 minutes of the reaction, NaOH was added to achieve a pH of 10. The reaction was allowed to continue on a magnetic stirrer for 2 hours. Thereafter, the suspension was centrifuged and washed several times with deionized water to lower the pH to 7. Finally, the particles were suspended in 10 mL of dimethylesulphoxide.

Characterization of Fe₃O₄ NPs

One milliliter of the stock suspension of Fe₃O₄ NPs was diluted in 10 mL complete minimum essential medium eagle. The hydrodynamic size and zeta potential were determined using Zetasizer Nano ZS.

Cytotoxicity assessment

The cytotoxic potential of Fe₃O₄ NPs was assessed in MCF-7 cells after 6 and 24 hours of treatment using MTT and neutral red uptake (NRU) assays as described by Mosmann⁶ and Borenfreund and Puerner, respectively.⁷

Cellular internalization of NPs

The internalization of Fe₃O₄ NPs in MCF-7 cells was assessed according to the method described in our earlier study.⁸

Cell cycle analysis

The effect of Fe₃O₄ NPs on cell cycle was assessed according to the method described in our earlier study.⁸

Results and discussion

The mean hydrodynamic size and zeta potential of synthesized Fe₃O₄ NPs were 98.19±1.0 nm and 36±2 mV, respectively. Flow cytometric analysis revealed a significant (P<0.05) increase in the internalization of Fe₃O₄ NPs in MCF-7 cells after 24 hours exposure at the two higher concentrations, as evident by an increase in the side scatter intensity (Figure 1).

Figure 1 Internalization of Fe3O4 NPs in MCF-7 cells using flow cytometry. Notes: Data are expressed as mean ± standard error of the mean from three independent experiments. *P<0.05, when compared with control. Abbreviations: NPs, nanoparticles; MCF-7, human breast adenocarcinoma cell line.

In the cytotoxicity assays, namely NRU and MTT, Fe₃O₄ NPs were found to be biocompatible as there was no significant increase in the NRU (88% at concentration 150 μM/mL) and a reduction (96%) in the mitochondrial succinate dehydrogenase activity was observed at the highest concentration after 24-hour exposure (Figures 2A and B).

Figure 2 Cytotoxicity of Fe3O4 NPs in MCF-7 cells. (A) NR uptake (%); (B) MTT reduction (%). Notes: The viability of the control cells was considered as 100%. Data are expressed as mean ± standard error of the mean from three independent experiments. Abbreviations: NPs, nanoparticles; MCF-7, human breast adenocarcinoma cell line; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NR, neutral red.

Additionally, no change in cell cycle progression was observed in Fe₃O₄ NPs-treated MCF-7 cells, after 24-hour exposure (Figure 3).

Figure 3 Effect of Fe3O4 NPs on cell cycle progression in MCF-7 cells (A) control (B–E) cells treated with different concentration of Fe3O4 NPs and (F) cells treated with 4 mM EMS. Abbreviations: EMS, ethylmethane sulfonate; NPs, nanoparticles; MCF-7, human breast adenocarcinoma cell line.

Conclusion

Our results demonstrated that Fe₃O₄ NPs synthesized using the safe-by-design approach showed no adverse effect on cells, as assessed by cytotoxicity assays and cell cycle analysis in MCF-7 cells, even though they are significantly internalized. Therefore, these NPs have a potential to be used as a carrier for targeted drug delivery.

Acknowledgments

The authors acknowledge the funding from the Centre for Nanotechnology Research and Application (CENTRA) by The Gujarat Institute for Chemical Technology (GICT); and the European Union Seventh Framework Programme (FP7/2007–2013) under grant agreement number 263147 (NanoValid – Development of reference methods for hazard identification, risk assessment and LCA of engineered nanomaterials).

Disclosure

The authors report no conflicts of interest in this work.

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