Preparation of Fe<sub>3</sub>O<sub>4</sub>/Reduced Graphene Oxide Nanocomposites with Good Dispersibility for Delivery of Paclitaxel

Zhang, Xiaojuan; Cai, Wenbin; Hao, Lingyun; Feng, Suli; Lin, Qing; Jiang, Wei

doi:https://doi.org/10.1155/2017/6702890

Journal of Nanomaterials

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Abstract Introduction Experimental Results and Discussion Conclusions Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2017 | Article ID 6702890 | https://doi.org/10.1155/2017/6702890

Preparation of Fe₃O₄/Reduced Graphene Oxide Nanocomposites with Good Dispersibility for Delivery of Paclitaxel

Xiaojuan Zhang,¹Wenbin Cai,¹Lingyun Hao,¹Suli Feng,¹Qing Lin,¹and Wei Jiang²

Academic Editor: Xuping Sun

Received13 Oct 2017

Accepted05 Dec 2017

Published31 Dec 2017

Abstract

The Fe₃O₄/reduced graphene oxide (Fe₃O₄/RGO) nanocomposites with good dispersibility were synthesized for targeted delivery of paclitaxel (PTX). Firstly, the superparamagnetic Fe₃O₄/functional GO nanocomposites were prepared via hydrothermal method in which GO sheets were modified by surfactant wrapping. The Fe₃O₄/RGO nanocomposites were successively prepared through the reduction of graphene oxide. The products were investigated by Fourier-transform infrared spectrum, X-ray diffraction, scanning electron microscopy, transmission electron microscopy, thermogravimetric analysis, and vibration sample magnetometry. It was found that spherical Fe₃O₄ nanoparticles were uniformly anchored over the RGO matrix and the nanocomposites were superparamagnetic with saturation magnetization (Ms) of 9.39 emu/g. Then PTX was loaded onto Fe₃O₄/RGO nanocomposites, and the drug loading capacity was 67.9%. Cell viability experiments performed on MCF-7 demonstrated that the Fe₃O₄/RGO-loaded PTX (Fe₃O₄/RGO/PTX) showed cytotoxicity to MCF-7, whereas the Fe₃O₄/RGO displayed no obvious cytotoxicity. The above results indicated that Fe₃O₄/RGO/PTX nanocomposites had potential application in tumor-targeted chemotherapy.

1. Introduction

Paclitaxel (PTX) is used for the first-line chemotherapeutics in breast cancer, lung cancer, and so on [1]. For improving the bioavailability of PTX, some groups made different carriers to load PTX such as hydrogels [2, 3], gel system [4], implantable chitosan film [5], nanogel [6], nanoparticles [7, 8], and nanostructured lipid carrier [9].

The graphene nanocomposites were studied for drug loading and delivery, since the hydrophobic drugs can be loaded on to graphene sheets by π-π stacking. For example, Angelopoulou et al. developed GO/PLA-PEG nanocomposites loading with PTX, which showed satisfactory high loading capacity, controlled release, and cytoxicity against A549 cancer cells [10]. Zhang et al. synthesized PEG modified nano graphene oxide for delivering PTX and indocyanine green (ICG), which had good fluorescence labeling and therapy effect [11].

Moreover, graphene is used as nanoscale building blocks for new nanocomposites because of its unique properties [12–14]. Recent research showed that magnetic nanoparticles (MNPs) could be attached to graphene, which can be applied in magnetic resonance imaging [15], targeted drug delivery [16, 17], biocompatible adsorbent [18], magnetic solid phase extraction [19], microwave electromagnetic [20], Schottky diode applications [21], and biomolecule immobilization [22]. Thus, the bifunctional graphene drug delivery system for magnetic targeting and drug loading was developed in this paper.

In this paper, the magnetic graphene oxide (Fe₃O₄/GO) for delivering PTX was prepared by hydrothermal method, and Fe₃O₄/GO was subsequently reduced to Fe₃O₄/RGO with good dispersibility. The Fe₃O₄/RGO nanocomposites were characterized, and the drug loading and release of PTX were also investigated. Furthermore, the inhibitory effect of Fe₃O₄/RGO-loaded PTX (Fe₃O₄/RGO/PTX) on MCF-7 cells was conducted.

2. Experimental

2.1. Materials

Iron(III) chloride hexahydrate (FeCl₃·6H₂O), Hexadecyl trimethyl ammonium bromide (CTAB), and Poly(sodium 4-styrenesulfonate) (PSS) were of analytical grade. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), PTX, and Triblock copolymers (PEO₉₉-PPO₆₇-PEO₉₉) PF127 were purchased from Sigma. The graphene oxide (GO) was purchased by Xian’feng Nanotechnologies Co. Ltd., China.

2.2. Synthesis and Application of Fe₃O₄/RGO/PTX Nanocomposites

The Fe₃O₄/RGO/PTX nanocomposites synthesis was shown in Figure 1.

2.2.1. Synthesis of Fe₃O₄/RGO/PTX Nanocomposites

To control the particle size of Fe₃O₄ nanoparticles, the GO was modified by the polymer wrapping technique, in which cationic CTAB (1 g) was adsorbed firstly on GO to create a charged template and anionic PSS (0.24 g) was subsequently grafted onto the surfaces of GO (as shown in Figure 1). The mixture of GO modified by CTAB and PSS was obtained, named functional GO (f-GO). The assembly of Fe₃O₄ nanoparticles on f-GO (Fe₃O₄/f-GO) was self-prepared using hydrothermal reaction [23]. For comparison, we synthesized Fe₃O₄/GO without modifying GO.

Secondly, PF127 (400 mg) was dropped to Fe₃O₄/f-GO solution (15 mL, 1.0 mg/mL) and hydrazine monohydrate (400 μL) was then added to reduce the GO [24]. After reaction, Fe₃O₄/RGO nanocomposites were obtained by pressure filtration. Lastly, the PTX was loaded by adding 0.01 g Fe₃O₄/RGO to PBS buffer solution containing PTX (1 mg/mL, 30 mL) at 25°C for 16 h in dark. The suspension was centrifuged and freeze-dried, and Fe₃O₄/RGO/PTX was obtained.

2.2.2. Drug Release of Fe₃O₄/RGO/PTX

The release of Fe₃O₄/RGO/PTX was tested in different buffer (7.4 and 5.0). 10 mg Fe₃O₄/RGO/PTX were suspended in 50 mL buffer at 37°C. The incubated solution (3 mL) was taken out and changed with equal volume buffer. The PTX release was characterized by UV–visible spectrophotometer (UV1101M054) (at 230 nm). The loading efficiency could be calculated by where is the weight of PTX in the Fe₃O₄/RGO/PTX and is the total weight of Fe₃O₄/RGO/PTX.

2.2.3. Cytotoxicity Assays

Cytotoxicity of free Fe₃O₄/RGO and Fe₃O₄/RGO/PTX was tested on MCF-7 cells. The MTT cell toxicity [25] was measured at 490 nm by enzyme-labeled Instrument (Multiskan FC, Thermo Scientific). Fe₃O₄/RGO and Fe₃O₄/RGO/PTX with different concentrations were added to cells group (six wells) for 24 h, respectively. The fluorescence microscopy of viable cells was characterized by Olympus CKX41.

2.3. Characterization

The structure was characterized by X-ray diffraction (Bruker D8, XRD). Bruker VECTOR 22 spectrometer was used to measure infrared spectrum (IR). The morphology was measured by scanning electron microscopy (Model S-4800, SEM) and transmission electron microscopy (Tecnai 12, TEM). Thermogravimetric analysis was characterized by thermal analyzer (TA2100, America TA). And vibration sample magnetometry (Lake Shore 7410, VSM) was used to detect the magnetic properties.

3. Results and Discussion

3.1. XRD Analysis

As exhibited in Figure 2, the diffraction peak of f-GO appeared at 22.9° which originated from the diffraction on its (002) layer planes [26]. The reflection peaks at 30.1°, 35.4°, 43.1°, 53.5°, 56.9°, and 62.5° can be assigned to (220), (311), (400), (422), (511), and (440) crystal planes of cubic magnetite [27]. It can be concluded that the magnetite-GO heterostructure was formed.

3.2. FT-IR Analysis

The FT-IR spectra for (a) GO, (b) Fe₃O₄/f-GO, (c) Fe₃O₄/RGO, and (d) Fe₃O₄/RGO/PTX were exhibited in Figure 3. In Figure 3(a), the C=O stretching vibration peaks appeared at 1725 cm⁻¹. The peak at 3430 cm⁻¹ was assigned to –OH bond, and the C=C bond was exhibited at 1617 cm⁻¹. In Figure 3(b), the Fe–O [28, 29] characteristic band appeared at 700 cm⁻¹. In addition, the bands at 2975 cm⁻¹ and 2890 cm⁻¹ (Figure 3(c)) could be ascribed to –CH₃ and –CH₂, which exhibited that Fe₃O₄/RGO was successfully modified by PF127. In Figure 3(d), the peaks at 1072, 1247, 707, 1652, and 1722 cm⁻¹ could be ascribed to the C–O ester, –COO, C–H aromatic, C=O amide, and C=O ester [30]. It can be concluded that PTX was loaded on Fe₃O₄/RGO/PTX.

3.3. SEM Analysis

Figure 4(a) showed that the GO had flake structure. After surfactant modification, the f-GO (Figure 4(b)) had good dispersibility. From Figure 4(c), before GO modification, few Fe₃O₄ nanoparticles were supported on the GO nanosheets. After GO modification, Figure 4(d) exhibited that spherical Fe₃O₄ nanoparticles were homogeneously distributed over the GO nanosheets, because the modified GO were covered with a negatively charged polyelectrolyte which can insure homogenous, high-density coverage of positively charged Fe³⁺ by electrostatic attraction. In Figure 4(e), after PF127 modification and reduction, Fe₃O₄ nanoparticles of 100 nm were loaded on the surface of RGO nanosheets. In addition, no free Fe₃O₄ nanoparticles were observed outside of RGO, indicating a strong interaction between Fe₃O₄ nanoparticles and RGO by modification by PF127. In Figure 4(f), it was observed that the Fe₃O₄/RGO/PTX had good morphology and dispersability, which could be applied in subsequent experiments.

(a)

(b)

(c)

(d)

(e)

(f)

3.4. TEM Analysis

Figure 5(a) showed TEM images of f-GO, which exhibited a typical wrinkled morphology and good dispersibility. In Figure 5(b), it was found that the GO carbon sheets were decorated by Fe₃O₄ nanoparticles with diameters from 50 nm to 100 nm. From the TEM of Fe₃O₄/f-GO nanocomposites with high resolution (Figure 5(c)), it was exhibited that Fe₃O₄ nanoparticles supported on GO nanosheets had good dispersibility and sphericity. After being modified by PF127 (Figure 5(c)), the Fe₃O₄/RGO nanocomposites had good dispersibility, from which it can be concluded that good dispersibility may be ascribed to the PF127 immobilization. After loading with PTX (Figure 5(d)), Fe₃O₄ nanoparticles were still firmly anchored on the surface of RGO.

3.5. TG Analysis

From Figure 6(a), 40.4% mass loss before 200°C was due to desorption of H₂O and pyrolysis of oxygen-containing group [24]. The pyrolysis of carbon skeleton was exhibited at 50.3% mass loss between 200 and 550°C. Figure 6(b) showed that 17.4% weight loss before 300°C was the removal of labile groups. A further 32.2% weight loss at 400°C (Figure 6(b)) was due to the decomposition of carboxyl group. From Figure 6(c), 3.6% weight loss below 200°C was corresponding to labile oxygen-containing functional groups. A further 32.3% weight loss was observed at around 400°C (Figure 6(c)), which was due to the decomposition of PF127. Finally, 43.1% at around 500°C was ascribed to the carbon skeleton pyrolysis.

(a)

(b)

(c)

3.6. VSM Analysis

In Figure 7(a), the products showed superparamagnetism without any hysteresis [31]. The saturation magnetization () of Fe₃O₄/f-GO was 9.39 emu/g. Before GO modification, the of as-obtained Fe₃O₄/GO nanocomposites decrease to 6.50 emu/g; it was due to the fewer Fe₃O₄ nanoparticles supported on the GO nanosheets. After drug loading, the of Fe₃O₄/RGO/PTX was 5.23 emu/g (Figure 7(c)). It could be seen that Fe₃O₄/RGO/PTX maintained good saturation magnetization, which was critical for further application.

3.7. UV Analysis

The release profiles of PTX from Fe₃O₄/RGO/PTX were shown in Figure 8. The standard curve of PTX was (2), and the drug loading efficiency of Fe₃O₄/RGO/PTX was 67.9%.

In Figure 8, a sustained PTX release at different pH values was observed from the Fe₃O₄/RGO/PTX. About 96.6% (Figure 8(a)) and 52.1% (Figure 8(b)) of PTX were released at pH = 5.5 and pH = 7.4 after 120 h. Moreover, the decreasing π-π stacking was the reason for rapid PTX release at pH = 5.5.

3.8. In Vitro Cytotoxicity Assay

In Figure 9(a), the free Fe₃O₄/RGO had no considerable cytotoxicity to MCF-7 cells. In Figure 9(b), with adding the Fe₃O₄/RGO/PTX, an obvious decrease in cell viability was observed. When the concentration of Fe₃O₄/RGO/PTX was 200 μg/mL, the cell viability was reduced to 31%. With adding the 500 μg/mL Fe₃O₄/RGO/PTX, the cell viability was rapidly reduced to 11%. It can be concluded that with increasing Fe₃O₄/RGO/PTX concentration the cell viability of MCF-7 decreased.

(a)

(b)

The cell viability results of products were also confirmed by fluorescence microscopy. The largest number of viable cells was exhibited in the control group (Figure 10(a)). With adding Fe₃O₄/RGO with different concentration (Figure 10(b), 1 μg/mL; Figure 10(c), 10 μg/mL; and Figure 10(d), 100 μg/mL), MCF-7 cells had no obvious morphological change.

(a)

(b)

(c)

(d)

The maximum viable cells were shown in the control group (Figure 11(a)). In Figure 11(b), with adding 100 μg/mL of Fe₃O₄/RGO/PTX, the number of viable MCF-7 cells reduced rapidly. When the concentration of PF127/Fe₃O₄/RGO/PTX increased to 200 and 500 μg/mL, few cells were observed (Figures 11(c) and 11(d)). It was confirmed that Fe₃O₄/RGO/PTX showed high antitumor activity for MCF-7 cells, which was ideal materials for tumor-targeted chemotherapy.

(a)

(b)

(c)

(d)

4. Conclusions

In conclusion, Fe₃O₄/RGO nanocomposites loading with PTX were successfully prepared. The method could be used for preparing other graphene-based nanocomposites. Moreover, the Fe₃O₄/RGO nanocomposites exhibited high drug loading efficiency and pH-dependent release, which was because of the π-π stacking and hydrophobic interactions between PTX and Fe₃O₄/RGO. With adding low concentration (200 μg/mL), the Fe₃O₄/RGO/PTX exhibited controlled release and inhibition of the growth of MCF-7 cells. Therefore, Fe₃O₄/RGO/PTX nanocomposites had potential application in tumor magnetically targeted therapy.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This work was supported by the sponsorship of Jiangsu Overseas Research & Training Program for University Prominent Young & Middle-Aged Teachers and Presidents and the research grants from the Natural Science Fund of Jiangsu Province (no. BK20130094).

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Copyright © 2017 Xiaojuan Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Journal of Nanomaterials

Preparation of Fe3O4/Reduced Graphene Oxide Nanocomposites with Good Dispersibility for Delivery of Paclitaxel

Abstract

1. Introduction

2. Experimental

2.1. Materials

2.2. Synthesis and Application of Fe3O4/RGO/PTX Nanocomposites

2.2.1. Synthesis of Fe3O4/RGO/PTX Nanocomposites

2.2.2. Drug Release of Fe3O4/RGO/PTX

2.2.3. Cytotoxicity Assays

2.3. Characterization

3. Results and Discussion

3.1. XRD Analysis

3.2. FT-IR Analysis

3.3. SEM Analysis

3.4. TEM Analysis

3.5. TG Analysis

3.6. VSM Analysis

3.7. UV Analysis

3.8. In Vitro Cytotoxicity Assay

4. Conclusions

Conflicts of Interest

Acknowledgments

References

Copyright

Preparation of Fe₃O₄/Reduced Graphene Oxide Nanocomposites with Good Dispersibility for Delivery of Paclitaxel

2.2. Synthesis and Application of Fe₃O₄/RGO/PTX Nanocomposites

2.2.1. Synthesis of Fe₃O₄/RGO/PTX Nanocomposites

2.2.2. Drug Release of Fe₃O₄/RGO/PTX