Graphene oxide coated Fe₃O₄@mSiO₂ NPs for magnetic controlled bioimaging

Abstract

A novel GO coated porous magnetic NPs (Fe₃O₄@mSiO₂@GO) for bioimaging was synthesized. First, Fe₃O₄ magnetic NPs were prepared through a solvothermal reaction. Second, through a surfactant-templating approach with cetyltrimethylammonium bromide (CTAB) as a template, a mesostructured CTAB/SiO₂ composite was deposited on Fe₃O₄ NPs surfaces. Third, CTAB templates were removed to form a mesoporous SiO₂ shell, resulting in mesoporous magnetic (Fe₃O₄@mSiO₂) NPs. Forth, the magnetic NPs surfaces were functionalized with amino groups by (3-aminopropyl)triethoxysilane (APTES). Finally, the dye rhodamine B (RB) was loaded inside porous magnetic NPs, and the magnetic NPs surfaces were then wrapped with graphene oxide (GO) nanosheets. SEM analysis revealed that GO sheets have been successfully coated on magnetic NPs surfaces. The hysteresis loops analysis indicated that these magnetic NPs still retained strongly magnetic property. Furthermore, Fe₃O₄@mSiO₂@GO NPs could efficiently protect the loaded dye from releasing. In addition, MTT assay revealed that the RB loaded Fe₃O₄@mSiO₂@GO NPs exhibited insignificant cytotoxicity at moderate concentrations and the RB loaded Fe₃O₄@mSiO₂@GO NPs could undergo target-directed move under the action of a magnetic field. The noncytotoxic magnetic hybrids presented significant potential for applications in cell imaging.

---

Introduction

Magnetic iron oxide NPs have attracted extensive research interest due to their unique physicochemical properties and great potential for various biomedical applications. In recent decades, magnetic NPs, especially Fe₃O₄ (magnetite) and Fe₂O₃ (maghemite), have been applied in various fields, particularly in biotechnology and biomedicine, such as enzyme-immobilization,¹ separation and purification,² biosensing and bioelectrocatalysis,³ magnetic resonance imaging,⁴ drug delivery,⁵ and tumor therapy.⁶ The requirements for any biomedical application of magnetic NPs include the chemical stability, biocompatibility and strong magnetization of the dispersed magnetic NPs. However, these magnetic particles are sensitive and unstable, especially under acidic conditions. In order to tackle these problems Fe₃O₄ particles are usually protected with proper shells to form core–shell structures such as Fe₃O₄@Al₂O₃ (ref. 7) and Fe₃O₄@SiO₂ (ref. 8) and so on. For example, the silica shell can not only prevent the aggregation of the Fe₃O₄ particles but also protect the inner magnetite core from oxidation, it also provides numerous surface Si–OH groups for further modification,⁹ especially mesoporous silica.

Mesoporous materials, which possess easily modified surface, ordered pores, proper pore volume and large surface area, are widely applied in various fields.^10–14 Since the first discovery in 1992,^15,16 synthesis and applications of mesoporous silica microspheres have attracted great interest due to their outstanding properties.^11,17 Up to now, mesoporous silica NPs have been well studied and employed as drug delivery platforms.^14,18 For example, Zhang¹⁹ et al. studied polymeric prodrug grafted hollow mesoporous silica nanoparticles for drug release. Liu²⁰ et al. prepared gold nanorods coated with mesoporous silica shell as drug delivery system, in this paper 1-tetradecanol molecules were also utilized as gatekeepers to regulate the drug release. However, most research work just focused on conventional constant, rather than controlling the drug release to the targeting sites. Undoubtedly, the combination of mesoporous silica and magnetic property can generate a new class of materials with great application potential.^21–26 The core–shell magnetic mesoporous silica NPs have strong magnetic responsivity, high dispersibility and orientated and accessible mesopores, which is highly valuable. On the other hand, the integration of mesoporous silica NPs and graphite oxide (GO) via electrostatic adsorption properties have been applied as an avenue for biological applications such as fluorescence imaging.²⁷ For example, Yang²⁸ et al. recently reported the fabrication of GO-encapsulated metal oxide NPs as high-performance anode materials through the co-assembly of GO sheets and positively charged oxide NPs. Zhao²⁷ et al. studied graphene oxide wrapping on squaraine-loaded mesoporous silica nanoparticles for bioimaging.

GO with its unique structure provides a broad range of applications, including DNA detection,^29,30 drug delivery,^31,32 and biological imaging.^33–36 GO produced by the oxidative treatment of graphite is two-dimensional and full of hydrophilic groups around the edges and on the planar surfaces, therefore, it has been an attractive choice as the substrate for preparation of nanocomposites due to its large surface area, novel optical, electronic, mechanical, and catalytic properties. The hydroxyl groups and carboxyl groups are responsible for the negative charges on the GO sheets in aqueous suspensions.^37,38 These functional groups will benefit for the preparation of GO composites with other materials either via electrostatic interactions^27,39,40 or covalent bonding.^39,41–43

Herein we synthesized a novel Fe₃O₄@mSiO₂@GO magnetic NPs with loaded RB for bioimaging. The magnetic Fe₃O₄ particles were prepared through a solvothermal reaction,⁴⁴ followed by the surface deposition of mesostructured CTAB/SiO₂ composite through a surfactant-templating approach with cetyltrimethylammonium bromide (CTAB) as template. The CTAB template was removed and then the magnetic NPs were aminated by APTES. The Fe₃O₄@mSiO₂ NPs were loaded with the dye RB, followed by wrapping of GO around the surfaces of Fe₃O₄@mSiO₂ NPs via electrostatic interaction. This novel magnetic hybrid material exhibited remarkable stability and could efficiently protect the loaded dye from releasing and could undergo target-directed move under the action of a magnetic field. Therefore, the mesoporous magnetic hybrids with superior properties present potential for application in cell imaging.

Experimental

Materials

Ferric chloride hexahydrate (FeCl₃·6H₂O, 99%) and ethylene glycol (AR) were purchased from Adamas-beta. Sodium citrate (99%), sodium acetate trihydrate (NaAc, 99%) and ethanol (AR) were purchased from Aldrich. Ammonia water (NH₃·H₂O, 28 wt%), hexadecyl trimethyl ammonium bromide (CTAB, 99%), tetraethyl orthosilicate (TEOS, AR), (3-aminopropyl)triethoxysilane (APTES, 99%), ammonium nitrate (99%) and rhodamine B (RB, 99%) were purchased from Aladdin. GO was prepared by a modified Hummer's method.^45,46

Instrumentation

The TEM images of the magnetic NPs were taken on a JEOL JEM-1200EX microscope at an accelerating voltage of 100 kV. The particles were dispersed in water (1 mg mL⁻¹) and deposited onto holey carbon grids (200 mesh copper holey carbon) and allowed to air-dry at room temperature. Fourier-transform infrared (FT-IR) spectroscopy was obtained using Tensor 27 produced by Bruker Corporation. Each sample was analyzed using 128 scans. The X-ray photoelectron spectroscopy (XPS) measurements were performed using a Kratos Axis Ultra DLD spectrometer employing a monochromated Al Kα X-ray source (h_γ = 1486.7 eV), hybrid (magnetic/electrostatic) optics, and a multi-channel plate, and delay line detector. Spectra were recorded at a takeoff angle of 35 (angle between the plane of the sample surface and the entrance lens of the analyzer) with pass energy of 150 eV. UV spectra were recorded on a UV/V-16/18 UV spectrophotometer (Shanghai Mapada), which was equipped with a double beam proportion monitoring optical system with a wavelength detection range from 200 to 800 nm. The images of Fe₃O₄@mSiO₂@GO were measured using SEM analysis (Zeiss Auriga). Zeta potential was measured by Malvern Nanosizer. The magnetic property was measured at room temperature using VSM (VSM7407, Lakeshore, USA). Polarization microscopic image of the magnetic nanohybrids was obtained using a Nikon Eclipse E600 microscope equipped with a Nikon DXN1200 digital camera and a Nikon Plan Fluor 50× objective (Nikon, Tokyo, Japan). The excitation laser was reflected by a polarized mirror to a high numerical aperture (NA) oil objective (50×) and focused to a diffraction limited spot (∼300 nm) on the sample surface.

Synthesis of Fe₃O₄ NPs

The magnetic particles were synthesized through a solvothermal reaction according to the reported method.⁴⁴ FeCl₃·6H₂O (1.087 g) was dissolved in ethylene glycol (40 mL) to form a clear solution, NaAc (3.6 g) was added in five minutes and stirred to dissolve, followed by the addition of sodium citrate (0.225 g). The resulting mixture was stirred vigorously for 30 min and then sealed in a Teflon-lined stainless-steel autoclave (50 mL capacity). The autoclave was heated to 200 °C and maintained for 10 h, and then allowed to cool to room temperature. The black products were washed several times with ethanol and water and then dried.

Preparation of Fe₃O₄@mSiO₂

Magnetic mesoporous silica NPs with CTAB (Fe₃O₄@CTAB/SiO₂) were synthesized referring to the previously published method.⁴⁷ Fe₃O₄ (0.1 g) NPs was homogeneously dispersed in a mixture of CTAB (0.3 g), ethanol (60 mL), deionized water (80 mL) and NH₃·H₂O solution (1 g). The obtained mixture was then homogenized by vigorously stirring for 30 min. Finally, TEOS (0.4 g) was added to the mixture with continuous stirring at room temperature. After reacting for 8 h, the product was collected with a magnet and washed several times with ethanol and deionized water and then dried. The Fe₃O₄@CTAB/SiO₂ (0.101 g) NPs was then dispersed in a mixture of isopropyl alcohol (100 mL) and APTES (1 mL) and then refluxed for 5 h to functionalize the magnetic NPs surfaces with amino groups. The purified product was dispersed in ethyl alcohol (120 mL), followed by the addition of ammonium nitrate (1.15 g) and refluxed for 1 h to remove the template CTAB. The Fe₃O₄@mSiO₂ NPs were washed several times with ethanol and deionized water and then dried for subsequent use.

Preparation of Fe₃O₄@mSiO₂@GO

Fe₃O₄@mSiO₂ (40 mg) NPs were homogeneously dispersed in a mixed solvent of ethanol (25 mL) and deionized water (30 mL), followed by the addition of RB (0.1 g) and stirred for 10 h at room temperature. To the resulting mixture was added GO suspension (5 mL, 0.5 mg mL⁻¹) under stirring for 12 h at room temperature. Finally, the product was washed several times with ethanol and deionized water and then dispersed in deionzied water for further use. In addition, the loading efficiency of RB was determined by UV-vis spectra.

Cytotoxicity and image study of magnetic NPs

Human corneal stromal cells were seeded in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and L-glutamine under 5% CO₂ atmosphere at 37 °C for 24 h prior to the cytotoxicity study. Cell density in a 96-well microplate was 10⁵ per well. Magnetic NPs were pretreated under high temperature and high pressure before introduction to the cell culture. After incubation, the viability was chosen as cytotoxicity parameter and determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.⁴⁸ MTT solution (5 mg mL⁻¹) was added to each well and further incubation for 4 h. The medium was then discarded and HeLa cells were washed with phosphate buffered saline (PBS). The relative cell viability (%) was calculated based on the following formula: (A_{of treated cells}/A_{of untreated cells}) × 100%.

HeLa cells were seeded in complete DMEM medium for 24 h before treated with RB loaded Fe₃O₄@mSiO₂@GO. Then RB loaded Fe₃O₄@mSiO₂@GO was added, after 12 h incubation, the medium was removed. Further incubation of cells for another 4 h, 8 h and 12 h in original medium without fluorescent nanoparticles was carried out. The free RB loaded cells were also imaged as the contrast group. HeLa cells were washed with phosphate buffered solution (PBS) (pH = 7.4) for three times to remove the free NPs in the culture dish. The cells treated with RB loaded magnetic NPs were observed under a florescence microscope.

Results and discussion

Preparation of Fe₃O₄, Fe₃O₄@CTAB/SiO₂, Fe₃O₄@mSiO₂ and Fe₃O₄@mSiO₂@GO magnetic NPs

The procedure to prepare Fe₃O₄@mSiO₂@GO NPs is shown in Scheme 1. First, the Fe₃O₄ magnetite particles were prepared by a solvothermal reduction method at 200 °C.⁴⁴ Second, through a surfactant-templating approach with CTAB as a template, mesoporous CTAB/SiO₂ shell was deposited on the Fe₃O₄ microsphere. Third, the template CTAB was easily removed by ammonium nitrate ethanol solution to form a mesoporous SiO₂ shell on Fe₃O₄ (Fe₃O₄@mSiO₂). Fourth, the Fe₃O₄@mSiO₂ NPs were functionalized with amino groups by covalent attachment of APTES. Subsequently, the Fe₃O₄@mSiO₂ NPs were loaded with RB, followed by wrapping of GO around the surface of Fe₃O₄@mSiO₂ NPs via electrostatic interaction, resulting in well-dispersed Fe₃O₄@mSiO₂@GO NPs.

Scheme 1 Schematic illustration of the synthesis of RB loaded Fe₃O₄@mSiO₂@GO NPs.

Characterization of the Fe₃O₄, Fe₃O₄@CTAB/SiO₂, Fe₃O₄@mSiO₂ and Fe₃O₄@mSiO₂@GO magnetic NPs

The particle size and morphology of Fe₃O₄, Fe₃O₄@mSiO₂, Fe₃O₄@mSiO₂@GO NPs are shown in Fig. 1. It can be observed that the Fe₃O₄ NPs were of well spherical structure with good mono-dispersibility and the average diameter was about 200 nm as shown in Fig. 1a. The surfactant templating process resulted in CTAB/SiO₂ shell coated on the Fe₃O₄ NPs surfaces. The subsequent treatment with ammonium nitrate by refluxing could remove CTAB templates, resulting in magnetic mesoporous NPs. As shown in Fig. 1b, the thick mesoporous SiO₂ shell was clearly seen to be uniformly coated on Fe₃O₄ NPs surfaces. GO was prepared from purified natural graphite and the average size of GO sheets was about 500 nm as shown in Fig. 1c. The SEM image of Fe₃O₄@mSiO₂@GO NPs recorded in gentle beam mode revealed the morphologies of GO loaded Fe₃O₄@mSiO₂, which confirmed the successful coating of GO on the surface of Fe₃O₄@mSiO₂ (Fig. 1d).

Fig. 1 TEM images of Fe₃O₄ (a), Fe₃O₄@mSiO₂ (b) and GO sheets (c), SEM image of Fe₃O₄@mSiO₂@GO NPs (d).

In order to further confirm the composition and structure of Fe₃O₄, Fe₃O₄@SiO₂ and Fe₃O₄@mSiO₂ NPs, FT-IR spectra were recorded (Fig. 2). From FT-IR spectrum of Fe₃O₄, the strong absorption peak at about 582 cm⁻¹ should be attributed to Fe–O vibration. The characteristic peaks at about 1635 and 1403 cm⁻¹ represent the O–H bending vibration and C–H stretching vibration, respectively. The typical peak at 1072 cm⁻¹ can be ascribed to Si–O asymmetric stretching vibration, which suggested that silica shell was successfully coated onto the surfaces of Fe₃O₄ NPs. The characteristic peaks of Fe₃O₄@CTAB/SiO₂ at 929 and 2851 cm⁻¹ should be contributed to C–H stretching vibration from CTAB. The peak at 3425 cm⁻¹ was resulted from the stretching vibration of hydroxyl groups. Moreover, the disappearance of C–H stretching vibration peaks in the FT-IR spectrum of Fe₃O₄@mSiO₂ suggested the complete removal of CTAB.

Fig. 2 FT-IR spectra of Fe₃O₄, Fe₃O₄@CTAB/SiO₂, Fe₃O₄@mSiO₂ NPs.

The surface charges of Fe₃O₄ NPs, GO sheets, Fe₃O₄@CTAB/SiO₂, Fe₃O₄@mSiO₂–NH₂ and Fe₃O₄@mSiO₂@GO NPs were examined using ζ potential measurement in deionized water, as shown in Table 1. Fe₃O₄ NPs showed negative ζ of −26.3 ± 3.6 because of the presence of hydroxy groups on the surfaces of Fe₃O₄ NPs, while CTAB/SiO₂ modified Fe₃O₄ NPs exhibited a positive ζ potential of 39.5 ± 4.6 mV due to the electro-negativity of the CTAB template, which testified the silica template has been successfully coated on Fe₃O₄ NPs. Fe₃O₄@mSiO₂–NH₂ showed a positive ζ potential of 38.1 ± 1.7 mV due to the introduction of amine groups on the Fe₃O₄@mSiO₂ NPs surfaces. Thus, the strong electrostatic interactions between the negative charged GO sheets and positively charged Fe₃O₄@mSiO₂–NH₂ led to the formation of Fe₃O₄@mSiO₂@GO NPs. In addition, the Fe₃O₄@mSiO₂@GO NPs showed a positive ζ potential of −13.0 ± 0.6 mV, confirming that GO sheets have been successfully coated on the surfaces of Fe₃O₄@mSiO₂–NH₂ NPs.

Table 1 The ζ potentials of Fe₃O₄ NPs, GO sheets, Fe₃O₄@CTAB/SiO₂ NPs, Fe₃O₄@mSiO₂–NH₂ NPs and Fe₃O₄@mSiO₂@GO NPs in deionized H₂O

Samples	Zeta (ζ) potential/mV
Fe3O4	−26.3 ± 3.6
GO	−79.1 ± 2.1
Fe3O4@CTAB/SiO2	39.5 ± 4.6
Fe3O4@mSiO2–NH2	38.1 ± 1.7
Fe3O4@mSiO2@GO	−13.0 ± 0.6

---

In addition to FT-IR spectra analysis, the Fe₃O₄ and Fe₃O₄@CTAB/SiO₂ magnetic NPs were also characterized by XPS. The spectrum exhibited elemental signals at 294.32, 532.05 and 980.07 eV, which confirmed the presence of C1s, O1s and CkLL respectively (Fig. 3a). For Fe₃O₄ NPs, the characteristic peaks at 711.82 and 724.89 eV represented Fe2p_3/2 and Fe2p_1/2 (Fig. 3c) from iron oxide. The data is consistent with the reported values of pure Fe₃O₄.⁴⁹ However, the characteristic peak of Fe2p_3/2 and Fe2p_1/2 disappeared in the spectrum of Fe₃O₄@CTAB/SiO₂ NPs as shown in Fig. 3b. The peaks at 102.65, 149.26 and 532.05 eV can be assigned to Si2p, Si2s (Fig. 3d) and O1s, respectively, indicating that silica shell was successfully formed on the Fe₃O₄ NPs surfaces.

Fig. 3 XPS broad scan spectra of Fe₃O₄ NPs (a) and Fe₃O₄@CTAB/SiO₂ NPs (b). XPS narrow scan spectra of Fe (2p) (c) and Si (2s, 2p) (d).

Characterization of RB loaded magnetic NPs

The Fe₃O₄@mSiO₂@GO and RB loaded Fe₃O₄@mSiO₂@GO NPs were uniformly dispersed in deionized water, and then the UV/vis spectra were measured. As shown in Fig. 4a the absorption spectrum of RB loaded Fe₃O₄@mSiO₂@GO NPs showed an absorbance peak at about 554 nm corresponding to the RB dye. Nevertheless, the Fe₃O₄@mSiO₂@GO NPs without RB did not show any obvious absorbance peak at around 554 nm under the same condition. The result indicated that the dye RB has been successfully loaded into the magnetic mesoporous NPs. The successful loading of RB into the Fe₃O₄@mSiO₂@GO NPs was also evidenced by polarization microscopy. As shown in Fig. 4b the red particles were clearly observed by polarization microscopy, further evidencing the RB loading. It is noteworthy that the polarization microscopy cannot visualize the single nano-sized particles and the observed are mostly particle aggregates.

Fig. 4 UV/vis spectra of Fe₃O₄@mSiO₂@GO NPs (black) and RB loaded Fe₃O₄@mSiO₂@GO NPs (red) in aqueous medium (a) and polarized light microscopy image of RB loaded Fe₃O₄@mSiO₂@GO NPs (b).

Magnetic characterization using a magnetometer indicated that the Fe₃O₄@mSiO₂ NPs had a magnetization saturation value of 39.5 emu g⁻¹, which was lower than that of Fe₃O₄ NPs (70.2 emu g⁻¹) as shown in Fig. 5a. The phenomenon might be related to the shielding of Fe₃O₄ NPs by the magnetic inactive layer, that is the mesoporous silica shell. However, the decrease of magnetization saturation value did not seriously affect the magnetic. The Fe₃O₄@mSiO₂ NPs could be accumulated within 8 s in aqueous solution when the magnetic field was applied and redispersed quickly once the magnetic field was removed, which suggested that these magnetic NPs possessed excellent magnetic and can be used for varied applications. UV/vis absorbance spectroscopy was also used to monitor the release of dye molecules from RB loaded Fe₃O₄@mSiO₂@GO NPs and RB loaded Fe₃O₄@mSiO₂ NPs. RB loaded Fe₃O₄@mSiO₂@GO NPs and RB loaded Fe₃O₄@mSiO₂ NPs were uniformly dispersed into deionized water and subject to dialysis using a 2000 cut-off membrane. The outer aqueous solution of the dialysis membrane was measured by UV/vis absorbance spectroscopy. As shown in Fig. 5b a marked reduction of the dye release was observed when GO sheets were coated on RB loaded Fe₃O₄@mSiO₂ NPs surfaces, compared with GO-free Fe₃O₄@mSiO₂ NPs. The result proved that the surface covered GO sheets could effectively protect the dye release from the mesoporous NPs. Hence, these kinds of magnetic hybrids could envision great potential for application in fluorescence imaging.

Fig. 5 The hysteresis loops of Fe₃O₄ NPs and Fe₃O₄@mSiO₂ NPs (inserted images were the photograph of magnetic response of Fe₃O₄@mSiO₂ to an external magnetic field at 298 K) (a) and the dye RB release from RB loaded Fe₃O₄@mSiO₂ NPs (black) and RB loaded Fe₃O₄@mSiO₂@GO NPs (red) in aqueous solution at each time point (b).

The surface area and porous nature of Fe₃O₄@mSiO₂ NPs were characterized by N₂ adsorption/desorption measurements as shown in Fig. 6a. A typical type-IV isotherm indicative of mesoporous structures was observed, with a Brunauer–Emmett–Teller (BET) surface area of 721.4 m² g⁻¹. The pore size distribution curve (Fig. 6a, inset) derived from the adsorption branch reveals that the sample has a mesopore size of 2.41 nm. As shown in Fig. 6b the absorption spectrum of RB loaded Fe₃O₄@mSiO₂@GO NPs and the free RB solution showed an absorbance peak at about 554 nm, corresponding to the RB dye. According to the UV/vis absorption of RB at 554 nm, the encapsulation efficiency of RB was calculated to be 21.08%.

Fig. 6 Nitrogen adsorption–desorption isotherms and pore size distribution profile (inset) of Fe₃O₄@mSiO₂ NPs (a) and fluorescence spectroscopy analysis of the immobilization of RB loaded porous magnetic NPs. The UV/vis spectra of the RB solutions separated from the mixture of porous magnetic NPs with RB and the free RB solution at the same concentration (b).

Cell test of RB loaded magnetic NPs

To examine the potential of RB loaded Fe₃O₄@mSiO₂@GO NPs for biological application, the cytotoxic effect of RB loaded Fe₃O₄@mSiO₂@GO NPs was evaluated using MTT viability assay with human corneal stromal cells for an incubation period of 48 h. Fig. 7a showed the cell viabilities of HeLa cells treated with RB loaded Fe₃O₄@mSiO₂@GO NPs for 12, 36 and 48 h at the same dye concentration range (0.005–50 μg mL⁻¹). For an incubation period of 36 h, the cell viability was about 90%. All samples showed high viability of more than 80% after 48 h treatment. These results indicated RB loaded Fe₃O₄@mSiO₂@GO NPs showed insignificant cytotoxicity at moderate concentrations. The fluorescence imaging studies were performed with HeLa cells. Fig. 7b–e showed the fluorescence microscopy images of HeLa cells treated with RB loaded Fe₃O₄@mSiO₂@GO NPs after incubation periods of 4 h, 8 h, 12 h and 24 h. It can be seen that the cells maintained their normal morphology and the red fluorescent intensity of the cells increased with the increasing incubation time. However, when the incubation time was 24 h, the red fluorescent intensity of the cells was the same to that after 12 h incubation, which indicated the cells completely swallowed Fe₃O₄@mSiO₂@GO NPs in 12 h.

Fig. 7 Cytotoxicity (a) and fluorescence microscopy images of cells incubated with RB loaded Fe₃O₄@mSiO₂@GO NPs for 4 h (b), 8 h (c), 12 h (d) and 24 h (e).

The fluorescence microscopy images of cells incubated with RB loaded Fe₃O₄@mSiO₂@GO NPs after 12 h incubation for another 4 h (a), 8 h (b) and 12 h (c) were investigated. As shown in Fig. 8a–c, the fluorescent intensity of cells was almost the same, which proved that the surface immobilized GO sheets could effectively prevent the dye releasing from the mesoporous NPs. However, the fluorescence microscopy images of the free RB loaded cells indicated that the free RB intruded into the nucleus, which further proved the surface immobilized GO sheets could effectively protect the dye in the mesoporous NPs. In a word, our investigations demonstrated the great potential of the novel magnetic hybrid for bioimaging applications.

Fig. 8 Fluorescence microscopy images of cells incubated with RB loaded Fe₃O₄@mSiO₂@GO NPs after 12 h incubation, followed by the removal of RB loaded Fe₃O₄@mSiO₂@GO NPs and then incubated with the normal medium for another 4 h (a), 8 h (b) and 12 h (c) and cells after 12 h incubation with free RB and then in normal medium for another 4 h (d).

Conclusions

We have successfully prepared novel Fe₃O₄@mSiO₂@GO NPs that can be utilized for magnetic controlled bioimaging. The preparation of Fe₃O₄@mSiO₂@GO NPs started from the synthesis of Fe₃O₄ NPs via a solvothermal method, followed by the formation of the porous silica shells from the hydrolysis and condensation reaction of TEOS in the presence of template CTAB. After the removal of CTAB and loading of RB inside porous magnetic NPs, the magnetic NPs surfaces were wrapped with graphene oxide (GO) sheets to prevent the self-releasing of the loaded stuff. The hysteresis loops indicated that these magnetic NPs still remained strong magnetism. Most importantly, these Fe₃O₄@mSiO₂@GO NPs could efficiently protect the loaded dye from releasing, which can be used as stable imaging reagents. In addition, the RB loaded Fe₃O₄@mSiO₂@GO NPs exhibited insignificant cytotoxicity at moderate concentrations and could undergo target-directed move under the action of a magnetic field. These novel magnetic hybrids are expected to serve as a platform for a variety of biological applications including cellular imaging.

Acknowledgements

This work was supported by Qingdao Innovation Leading Talent Program, Natural Science Foundation of Qingdao (12-1-4-2-2-jch) and Taishan Scholars Program.

Notes and references

1. J. R. McCarthy and R. Weissleder, Adv. Drug Delivery Rev., 2008, 60, 1241–1251.
2. H.-Y. Park, M. J. Schadt, L. Y. Wang, I. I. S. Lim, P. N. Njoki, S. H. Kim, M.-Y. Jang, J. Luo and C.-J. Zhong, Langmuir, 2007, 23, 9050–9056.
3. R. De Palma, C. Liu, F. Barbagini, G. Reekmans, K. Bonroy, W. Laureyn, G. Borghs and G. Maes, J. Phys. Chem. C, 2007, 111, 12227–12235.
4. C. Corot, P. Robert, J.-M. Idée and M. Port, Adv. Drug Delivery Rev., 2006, 58, 1471–1504.
5. B. Chertok, A. E. David, B. A. Moffat and V. C. Yang, Biomaterials, 2009, 30, 6780–6787.
6. Z. Liu, Y. Liu, K. Yao, Z. Ding, J. Tao and X. Wang, J. Mater. Synth. Process., 2002, 10, 83–87.
7. A. Ballesteros-Gómez and S. Rubio, Anal. Chem., 2009, 81, 9012–9020.
8. Y.-H. Deng, C.-C. Wang, J.-H. Hu, W.-L. Yang and S.-K. Fu, Colloids Surf., A, 2005, 262, 87–93.
9. A.-L. Morel, S. I. Nikitenko, K. Gionnet, A. Wattiaux, J. Lai-Kee-Him, C. Labrugere, B. Chevalier, G. Deleris, C. Petibois and A. Brisson, ACS Nano, 2008, 2, 847–856.
10. J. Y. Ying, C. P. Mehnert and M. S. Wong, Angew. Chem., Int. Ed., 1999, 38, 56–77.
11. M. Vallet-Regí, F. Balas and D. Arcos, Angew. Chem., Int. Ed., 2007, 46, 7548–7558.
12. L. Giraldo, B. López, L. Pérez, S. Urrego, L. Sierra and M. Mesa, Macromol. Symp., 2007, 258, 129–141.
13. Z. Wu and D. Zhao, Chem. Commun., 2011, 47, 3332–3338.
14. A. Popat, S. B. Hartono, F. Stahr, J. Liu, S. Z. Qiao and G. Q. M. Lu, Nanoscale, 2011, 3, 2801–2818.
15. J. Beck, J. Vartuli, W. Roth, M. Leonowicz, C. Kresge, K. Schmitt, C. Chu, D. H. Olson and E. Sheppard, J. Am. Chem. Soc., 1992, 114, 10834–10843.
16. C. Kresge, M. Leonowicz, W. Roth, J. Vartuli and J. Beck, Nature, 1992, 359, 710–712.
17. I. I. Slowing, B. G. Trewyn, S. Giri and V. Y. Lin, Adv. Funct. Mater., 2007, 17, 1225–1236.
18. M. Manzano and M. Vallet-Regí, J. Mater. Chem., 2010, 20, 5593–5604.
19. Y. Zhang, C. Y. Ang, M. Li, S. Y. Tan, Q. Qu and Y. Zhao, ACS Appl. Mater. Interfaces, 2016, 8, 6869–6879.
20. J. Liu, C. Detrembleur, D. Pauw-Gillet, S. Mornet, C. Jérôme and E. Duguet, Small, 2015, 11, 2323–2332.
21. I. S. Ibarra, J. A. Rodriguez, J. M. Miranda, M. Vega and E. Barrado, J. Chromatogr. A, 2011, 1218, 2196–2202.
22. S. Liu, H. Chen, X. Lu, C. Deng, X. Zhang and P. Yang, Angew. Chem., 2010, 122, 7719–7723.
23. Q. Gao, D. Luo, J. Ding and Y.-Q. Feng, J. Chromatogr. A, 2010, 1217, 5602–5609.
24. J.-H. Wu, X.-S. Li, Y. Zhao, Q. Gao, L. Guo and Y.-Q. Feng, Chem. Commun., 2010, 46, 9031–9033.
25. P. Wu, J. Zhu and Z. Xu, Adv. Funct. Mater., 2004, 14, 345–351.
26. L. Zhang, S. Qiao, Y. Jin, Z. Chen, H. Gu and G. Q. Lu, Adv. Mater., 2008, 20, 805–809.
27. S. Sreejith, X. Ma and Y. Zhao, J. Am. Chem. Soc., 2012, 134, 17346–17349.
28. S. Yang, X. Feng, S. Ivanovici and K. Müllen, Angew. Chem., Int. Ed., 2010, 49, 8408–8411.
29. S. Bi, T. Zhao and B. Luo, Chem. Commun., 2012, 48, 106–108.
30. M. Luo, X. Chen, G. Zhou, X. Xiang, L. Chen, X. Ji and Z. He, Chem. Commun., 2012, 48, 1126–1128.
31. D. Depan, J. Shah and R. D. K. Misra, Mater. Sci. Eng., C, 2011, 31, 1305–1312.
32. H. Shen, L. Zhang, M. Liu and Z. Zhang, Theranostics, 2012, 2, 283.
33. Y. Wang, Z. Li, J. Wang, J. Li and Y. Lin, Trends Biotechnol., 2011, 29, 205–212.
34. L. Feng, L. Wu and X. Qu, Adv. Mater., 2013, 25, 168–186.
35. G. Gonçalves, M. Vila, M. T. Portolés, M. Vallet-Regi, J. Gracio and P. A. A. Marques, Adv. Healthcare Mater., 2013, 2, 1072–1090.
36. K. Yang, L. Feng, X. Shi and Z. Liu, Chem. Soc. Rev., 2013, 42, 530–547.
37. W. Scholz and H. Boehm, Z. Anorg. Allg. Chem., 1969, 369, 327–340.
38. A. Lerf, H. He, M. Forster and J. Klinowski, J. Phys. Chem. B, 1998, 102, 4477–4482.
39. Z. Liu, J. Liu, D. Li, P. S. Francis, N. W. Barnett, C. J. Barrow and W. Yang, Chem. Commun., 2015, 51, 10969–10972.
40. H. Yan, X. Tao, Z. Yang, K. Li, H. Yang, A. Li and R. Cheng, J. Hazard. Mater., 2014, 268, 191–198.
41. N. Kong, J. Liu, Q. Kong, R. Wang, C. J. Barrow and W. Yang, Sens. Actuators, B, 2013, 178, 426–433.
42. J. Liu, N. Kong, A. Li, X. Luo, L. Cui, R. Wang and S. Feng, Analyst, 2013, 138, 2567–2575.
43. Y. Shan, X. Y. Xu, K. Z. Chen and L. Gao, Adv. Mater. Res., 2013, 774–776, 532–535.
44. J. Liu, Z. Sun, Y. Deng, Y. Zou, C. Li, X. Guo, L. Xiong, Y. Gao, F. Li and D. Zhao, Angew. Chem., Int. Ed., 2009, 48, 5875–5879.
45. D. A. Dikin, S. Stankovich, E. J. Zimney, R. D. Piner, G. H. B. Dommett, G. Evmenenko, S. T. Nguyen and R. S. Ruoff, Nature, 2007, 448, 457–460.
46. W. S. Hummers and R. E. Offeman, J. Am. Chem. Soc., 1958, 80, 1339.
47. Y. Deng, D. Qi, C. Deng, X. Zhang and D. Zhao, J. Am. Chem. Soc., 2008, 130, 28–29.
48. P. Cerrai, G. Guerra, L. Lelli, M. Tricoli, R. S. Del Guerra, M. Cascone and P. Giusti, J. Mater. Sci.: Mater. Med., 1994, 5, 33–39.
49. G. Du, Z. Liu, X. Xia, Q. Chu and S. Zhang, J. Sol-Gel Sci. Technol., 2006, 39, 285–291.

---