Fullerene C₆₀ functionalized γ-Fe₂O₃ magnetic nanoparticle: Synthesis, characterization, and biomedical applications

Abstract

Hybrid magnetic nanoparticles composed from C₆₀ fullerene and γ-Fe₂O₃ were synthesized by hydrothermal method. XRD, FT-IR, VSM, SEM, and HR-TEM were employed for characterizations. The magnetic saturation value of C₆₀-γ-Fe₂O₃ magnetic nanoparticles was 66.5 emu g^{− 1}. Concentration of Fe in nanoparticles asdetermined by ICP-OES was 40.7% Fe. Particle size of C₆₀-γ-Fe₂O₃ magnetic nanoparticles was smaller than 10 nm. Maximum adsorption capacity of C₆₀-γ-Fe₂O₃ for flurbiprofen, a non-steroidal anti-inflammatory drug, was calculated from Langmuir isotherm as 142.9 mg g^{− 1}.

Keywords::

• biomedical application
• chemical synthesis
• magnetic materials
• magnetic properties

Introduction

Magnetic nanoparticles (MNPs) show magnetic behavior when exposed to magnetic fields and they do not retain any magnetism once the applied magnetic field is removed. These particles have an important application area in biotechnology (Gao et al. 2009). Metallic, bimetallic, and superparamagnetic iron oxide nanoparticles (SPIONs) are classified as MNPs (Veiseh et al. 2010). Among SPIONs, γ-Fe₂O₃ and Fe₃O₄ attained special attention due to their biocompatibilities, biodegradabilities, availabilities, stabilities, and high magnetic susceptibilities (Donadel et al. 2008, Yiu and Keane 2012). Magnetic nanoparticles have been widely used for protein adsorption (Liu et al. 2004), bacterial detection and protein purification (Gao et al. 2009), targeted drug delivery (Berry and Curtis 2003), cancer diagnosis/therapy (Kievit and Zhang 2011, Yigit et al. 2012), photodynamic therapy (Huang et al. 2011), bioanalytical sensor (Stanciu et al. 2009), as supporting materials for enzymes (Netto et al. 2013). SPIONs sized between 10 and 100 nm can be used for in vivo and in vitro studies due to size similarities with biological macromolecules, cells, and enzymes (Yiu and Keane 2012). Magnetic nanoparticles could be converted to biocompatible forms by coating with poly(ethyleneglycol), dextran, chitosan, copolymers, polyethyleneimine, liposomes, and micelles for in vivo studies (Veiseh et al. 2010, Yigit et al. 2012). Additionally, surface coating could also significantly influence the cytotoxicities of SPIONs (Donadel et al. 2008). Surface coating increases the half-life of SPIONs in blood by reducing the adsorption of proteins onto the surfaces (Gupta and Wells 2004).

New carbon allotrope containing 60 perfectly symmetrically arranged carbon atoms (C₆₀) was discovered in 1985 and called as buckminsterfullerene (Kroto et al. 1985). The combination of the unique three-dimensional shape, physical, chemical, electrical, and optical properties with the extremely rich carbon chemistry makes fullerene one of the most exciting materials of nanobiotechnology that this field produces and investigates the nanomaterials in biotechnology and medicine. Diameter of a fullerene C₆₀ molecule is 7 Å and this size makes it a potential nanomaterial for biological applications (Partha and Conyers 2009). Additionally, covalent functionalization of fullerene opens the door to various applications in the drug delivery (Brettreich et al. 2000), photodynamic cancer therapy (Mroz et al. 2007), gene delivery (Nakamura et al. 2000), antioxidant (Enes et al. 2009), magnetic resonance imaging (MRI) contrast agent (Tóth et al. 2005), and photocatalyst (Meng et al. 2012) fields. The role of functionalized fullerene in a new emerging research area, nanobiotechnology, that includes nanomedicine and biomedical applications was reviewed with details (Satoh and Takayanagi 2006, Partha and Conyers 2009). It should be noted that toxicity of functionalized fullerene is not well understood (Zhang et al. 2009). Self-assembled spherical nanostructures derived from amphiphilic fullerene were used as nanocarrier called as buckysomes for paclitaxel, a highly hydrophobic anticancer drug. It was reported that paclitaxel-embedded buckysomes demonstrated a similar efficacy to that of commerical one in cell viability studies (Partha et al. 2008). Antioxidant effects and in vivo radioprotective effect of a water-soluble antioxidant based on the hollow nanostructure of fullerenes derivative (dendrofullerene) containing 18 carboxylic groups was evaluated (Daroczi et al. 2006). A fullerene derivative bearing two diamino side chains bound to a plasmid vector DNA was used for nonviral gene delivery (Isobe et al. 2006). Due to its low stability and self-aggregation tendency, functionalization of fullerene is required. The water-soluble endohedral gadofullerene derivatives have found application area in MRI as contrast agent (Tóth et al. 2005).

The profen family including flurbiprofen {(R,S)-[2-(2-fluoro-4-phenyl) phenyl] propionic acid} is a member of non-steroidal anti-inflammatory (NSAI) drugs (NSAIDs) (Bae et al. 2006). Pharmacological activities of the commonly consumed NSAIDs are predominantly attributed to inhibitory effect on the activity of the cyclooxygenase (COX) enzyme catalyzing the first step in the conversion of arachidonic acid to prostaglandins and thromboxanes (Sorge et al. 1998, Hinz et al. 2001). Among available NSAI agents, flurbiprofen is the most commonly used one among others (Pignatello et al. 2002). The family exists in two stereo-isomeric forms, yet only an (S)-enantiomer exhibits pharmacological activity through the inhibition of the cyclooxygenase system while an (R)-enantiomer is not only biologically inactive but also demonstrates gastrointestinal toxicity and chiral inversion (Lee et al. 2003, Bae et al. 2006).

Therefore, new hybrid materials composed of fullerene and iron oxide would be extremely attractive. Unique properties of C₆₀ and magnetic nanoparticles could be combined to increase their activities and potential biomedical applications by further investigations. In this paper, C₆₀-γ-Fe₂O₃ magnetic nanoparticle was synthesized and magnetic properties, chemical composition, surface funtionalities, morphology, and micro structures of the synthesized magnetic nanoparticles were thoroughly characterized by SEM, TEM, FT-IR, ICP-OES, and vibrating sample magnetometer (VSM).

Materials and methods

Reagents and standards

Fullerene C₆₀ (> 98%), toluene, NH₄OH, flurbiprofen were supplied from Sigma-Aldrich (St. Loius, MO). FeCl₃·6H₂O and FeCl₂ were bought from Merck (Darmstadt, Germany). All chemicals were of analytical reagent-grade. Doubly distilled water was used throughout the experiments.

Instrumentation

Total Fe concentrations in C₆₀ conjugated γ-Fe₂O₃ magnetic nanoparticle and C₆₀ were determined by ICP-OES (Perkin Elmer, Optima 2100 DV) at the wavelength of 238.204 nm. Instrumentation conditions were given in our previous study (Kilinc and Aydin 2012). C₆₀ residue in synthesized SPIONs was monitored by UV-VIS spectrophotometer (Perkin Elmer, Lambda 25). Infrared spectra of magnetic nanoparticle in KBr pellet were recorded in the ranges of 4000–400 cm^{− 1} by Mattson Model 1000 FT-IR spectrophotometer. The Model P525 VSM measurement system (Quantum Design) for the physical property measurement system (PPMS) was used at 27°C. SEM analysis was carried out by LEO-Evo 40 XVP scanning electron microscope. HR-TEM images were recorded on Jeol JEM 2100F HRTEM instrument working at 200 kV with a probe size under 0.5 nm. Concentration of flurbiprofen was also measured by UV-VIS spectrophotometer at 250 nm (Kilinc and Aydin 2009).

Synthesis of γ-Fe₂O₃ SPIONs

Approximately 0.01 g of MNP and 0.03 g of fullerene C₆₀ were weighted and placed in a beaker. Three mL of concentrated HCl and 1.0 mL of HNO₃ and 0.5 mL of H₂O₂ were added and heated until dried. Residues were dissolved in 50 mL and 5.0 mL of 1.0 M HNO₃, for C₆₀ conjugated γ-Fe₂O₃ SPIONs and C₆₀, respectively. Total Fe concentrations were determined by ICP-OES. Surface functionalities were discussed by comparing their FT-IR spectra. The samples were prepared as film on KBr windows. The spectra were recorded in the transmission mode. Chemical structure of γ-Fe₂O₃ nanoparticle functionalized with fullerene C₆₀ was determined by XRD. XRD traces were recorded from 2Ө of 2°–80° with a 0.02° step size. VSM was employed to determine the magnetic saturation value of C₆₀-γ-Fe₂O₃ magnetic nanoparticles at room temperature. SEM was used for characterization of macrostructure of nanomaterials with different resolutions. The morphology and microstructure of C₆₀-γ-Fe₂O₃ magnetic nanoparticles were investigated by HR-TEM.

Synthesis of C₆₀-γ-Fe₂O₃ SPIONs

γ-Fe₂O₃ (0.36 g) was added to the solution of 0.17 g of C₆₀ fullerene dissolved in 100.0 mL of toluene. The mixture was sonicated for 5.0 min at 30°C and vigorously stirred for 3 days at room temperature. FeCl₃.6H₂O and FeCl₂, at the molar ratio of 2:1, were dissolved in distilled water and stirred in a 100.0 mL three necked flask. 30.0 mL of 5% NH₄OH solution was added dropwise at 75°C with vigorous stirring for about 2.0 h under nitrogen purge. It was subsequently washed with distilled water, toluene, and absolute ethanol until the C₆₀ absorption peak disappeared by monitoring UV-VIS spectra at 554 nm.

Adsorption of flurbiprofen on C₆₀-γ-Fe₂O₃

Adsorption of flurbiprofen on C₆₀-γ-Fe₂O₃ SPIONs was investigated. Briefly, flurbiprofen solutions at the concentration of 5.0 μg mL^{− 1} were prepared in water and buffer. Twenty mg of C₆₀-γ-Fe₂O₃ SPIONs was added to it and magnetically stirred for 30 min at 100 rpm. Experiments were performed at 25°C. Then, SPIONs were removed by a strong magnet. The concentrations of flurbiprofen in remaining solutions were determined by UV-VIS spectrophotometer at 250 nm. flurbiprofen adsorbed on C₆₀-γ-Fe₂O₃ SPIONs was also released with different solvent and buffers.

Results and discussion

Characterization of C₆₀ functionalized γ-Fe₂O₃ magnetic nanoparticles

Wide ranges of physical and chemical characterization are utilized in synthesizing and determining the functions of nanoparticles. Surface functionality, magnetization value and surface morphology are properties that should be considered and evaluated. Thus, C₆₀-γ-Fe₂O₃ magnetic nanoparticles were characterized by various physical and chemical techniques.

Fe concentrations in fullerene C₆₀, C₆₀-γ-Fe₂O₃ magnetic nanoparticles were determined by ICP-OES after wet digestion on hot plate by using concentrated HCl followed by mixture of HNO₃ and H₂O₂ (1:1, v/v). Fe concentration in pristine C₆₀ was 847.5 mg kg^{− 1} while in C₆₀-γ-Fe₂O₃ magnetic nanoparticles it was 40.8%. While Fe amount in pure γ-Fe₂O₃ was considered as 70%, it could be concluded C₆₀ reacted with γ-Fe₂O₃ to give a new product of γ-Fe₂O₃ coated with C₆₀. These also confirm the coating of C₆₀ on γ-Fe₂O₃.

Comparison of FT-IR spectra of γ-Fe₂O₃ magnetic nanoparticle, C₆₀ and C₆₀-γ-Fe₂O₃ magnetic nanoparticles is presented in Figure 1. The wide absorption band at about 3400 cm^{− 1} assigned to the O-H stretching vibration indicated that there were still some -OH groups that had not yet reacted on the surface of the γ-Fe₂O₃ nanoparticles. The peaks at about 630, 590, and 440 cm^{− 1} of γ-Fe₂O₃ corresponded to the metal-oxygen vibrational modes of the spinel compounds and are in agreement with the data in the literature (Pereira et al. 2010, Ganachari et al. 2012). Peaks at about 525, 575, 1180, and 1426 cm^{− 1} region corresponded to fullerene peaks (Mukhopadhyay et al. 2001). The distinct band 1426 cm^{− 1} in Figure 1b. was assigned to C = C vibrations and the band at 1170 cm^{− 1} was attributed to C-H in-plane bending vibrations (Barszcz et al. 2007). The peaks at approximately 1400 cm^{− 1} and 1600 cm^{− 1} observed in curves (a), (b), and (c) indicated a complex reaction between hydroxyl groups on the surface of magnetic nanoparticles. The clarity of the FTIR spectra and the absence of characteristic peaks of C₆₀ and γ-Fe₂O₃ were evaluated as coating of γ-Fe₂O₃ with C₆₀ as demonstrated in Figure 1c.

Figure 1. Comparison of FT-IR spectra of a) γ-Fe₂O₃ magnetic nanoparticle, b) C₆₀, c) C₆₀-γ-Fe₂O₃ SPIONs.

XRD patterns of C₆₀-γ-Fe₂O₃ magnetic nanoparticles are presented in Figure 2. The XRD peaks of the C₆₀-γ-Fe₂O₃ were compared with those reported in the literature and spectral library of instrument (Maghemite-Q, Card No. 25-1402). A series of characteristic peaks at 2θ = 30.5°, 35.8°, 43.8°, 54.2°, 57.7°, and 63.2°, correspond to (220), (311), (400), (422), (511), and (440) Bragg reflection, respectively. These were in agreement with standard maghemite (γ-Fe₂O₃) XRD patterns (Reddy et al. 2002). However, it was difficult to distinguish the two phases simply from XRD patterns since the XRD patterns of γ -Fe₂O₃ and Fe₃O₄ are reportedly very similar (Ganachari et al. 2012). Peaks at 2θ = 14.5°, 18.2°, and 27.4° correspond to pristine C₆₀. The data are in good agreement with that reported in literature (Oh et al. 2007, Baker et al. 2008, Sathish and Miyazawa 2012). These XRD data seem to confirm the coating of γ-Fe₂O₃ with C₆₀.

Figure 2. XRD patterns of C₆₀-γ-Fe₂O₃ SPIONs, A: peaks correspond to C₆₀, B: peaks correspond to γ-Fe₂O_3.

The microstructure and morphology of C₆₀-γ-Fe₂O₃ magnetic nanoparticles were evaluated by SEM and HR-TEM images. SEM images of C₆₀-γ-Fe₂O₃ magnetic nanoparticles are demonstrated on different resolutions in Figure 3 and the surface morphology analysis demonstrated that it had uniform size distribution. According to the SEM images, the agglomeration was strong in the bare γ-Fe₂O₃ nanoparticles. The agglomeration could be due to the Van der Waals force between the particles and the moisture in sample. Nearly spherical nanoparticles were observed from SEM images. TEM images of C₆₀-γ-Fe₂O₃ magnetic nanoparticles at different resolutions are presented in Figure 4. The main nearly spherical structures of SPIONs are clearly seen. From the TEM images it is clearly observed that particle size is lower than 10 nm. It should be noted that particle size of C₆₀-γ-Fe₂O₃ magnetic nanoparticles makes them usable and removable in vivo through extravasation and renal clearance (Gupta and Wells 2004). C₆₀-γ-Fe₂O₃ SPIONs present further functionalization through the targeted drug delivery and specific applications.

Figure 3. Comparison of SEM images of C₆₀-γ-Fe₂O₃ SPIONs.

Figure 4. Comparison of TEM and HR-TEM images of C₆₀-γ-Fe₂O₃ SPIONs.

The magnetic properties of magnetic nanoparticles were analyzed by VSM. Magnetization curve is presented in Figure 5. The magnetic saturation value of C₆₀-γ-Fe₂O₃ was equal to 66.5 emu g^{− 1} which was significantly lower than that for the reported multidomain bulk particles (74 emu g^{− 1}) (Shafi et al. 2002). This was attributed to decrease in particle size from 100 nm to < 20 nm ranges and to the coating on surface of γ-Fe₂O₃ with C₆₀ (Morales et al. 1997). The result revealed no remanence effect, reflecting superparamagnetic behavior of C₆₀-γ-Fe₂O₃. Additionally, it is clear from high magnetic saturation value that synthesized SPIONs could be useful for biomedical application. Taken together, these data confirm that magnetic nanoparticles composed of γ-Fe₂O₃-C₆₀ SPIONs were successfully prepared.

Figure 5. VSM magnetization curve of C₆₀-γ-Fe₂O₃ SPIONs.

Adsorption of flurbiprofen on C₆₀-γ-Fe₂O₃

Effect of pH on the adsorption on C₆₀-γ-Fe₂O₃ was investigated in the pH range of 4.0–8.0. Fifty mL of flurbiprofen solution at 5.0 μg mL^{− 1} concentration was added to 20.0 mg of C₆₀-γ-Fe₂O₃. pH of the aqueous solutions were adjusted to desired value by adding NaOH and HNO₃. After adsorption, SPIONs were magnetically removed. Maximum adsorption was observed at approximately pH 6.0. Results are presented in Figure 6. Effect of amount of SPIONs was also investigated in the ranges of 5.0–50.0 mg of C₆₀-γ-Fe₂O₃. As shown in Figure 7, higher adsorption values were achieved by lowering in SPIONs amount. Effect of initial concentration of flurbiprofen on the adsorption was investigated in the concentration ranges of 1.0–75.0 μg mL^{− 1} of flurbiprofen. As shown in Figure 8 saturation on the surface of SPIONs was observed at approximately 50 μg mL^{− 1} of flurbiprofen. Results from adsorption experiments were applied to Langmuir and Freundlich isotherms. By considering the correlation coefficients, it could be said that adsorption results well fitted to Langmuir isotherm. From the plot of c_eq/q_eq (where c_eq: equilibrium flurbiprofen concentration, q_eq: mg adsorbed flurbiprofen on per gram of C₆₀-γ-Fe₂O₃) to c_eq, equation of curve was obtained as y = 0.007x + 0.0335, with coefficient of r² = 0.9922. From Langmuir equation, as, maximum adsorption capacity was 142.9 mg g^{− 1} while K_b was 0.208 L mg^{− 1}, it could be concluded that C₆₀-γ-Fe₂O₃ had high surface area. Monolayer adsorption of flurbiprofen on SPIONs could be valid for adsorption of flurbiprofen on C₆₀-γ-Fe₂O₃. Maximum adsorption capacity of C₆₀-γ-Fe₂O₃ was calculated as 142.9 mg g^{− 1} (pH of the solution was 6.0, 20.0 mg of C₆₀-γ-Fe₂O₃) from Langmuir isotherm.

Figure 6. Effect of pH on adsorption of flurbiprofen on C₆₀-γ-Fe₂O₃ SPIONs.

Figure 7. Effect of amount of C₆₀-γ-Fe₂O₃ SPIONs on the adsorption of flurbiprofen.

Figure 8. Effect of initial concentration of flurbiprofen on the adsorp-tion on C₆₀-γ-Fe₂O₃ SPIONs on the adsorption of flurbiprofen.

Conclusion

The aim of this study was to synthesize and characterize the C₆₀ and iron oxide based magnetic nanoparticles with possible application in nanobiotechnology. Secondly, its application in analytical and bioanalytical studies was also investigated. Limitations in the structural and biological compatibility of SPIONs could be overcome by chemical functionalization with carbon based materials such as fullerene. FT-IR, VSM, XRD, SEM, and TEM data confirmed the structure and nanocrystalline nature of the synthesized magnetic particles. The results obtained from this study were expected to give an insight for synthesis and application of C₆₀-γ-Fe₂O₃ magnetic nanoparticles in nanobiotechnology and nanomedicine through in vitro and in vivo studies. It was observed that synthesized SPIONs were nearly completely spherical structure that contains γ-Fe₂O₃ core C₆₀ shell. Adsorption of flurbiprofen on C₆₀-γ-Fe₂O₃ was examined with details. It was found that monolayer adsorption of drug on SPIONs was valid from Langmuir isotherms. By considering the potential applications of C₆₀ functionalized SPIONs, further investigations are needed to investigate the applicability, toxicity etc.

Declaration of interest

The author reports no declaration of interest. The author alone is responsible for the content and writing of the paper.

The present work was carried out under the financial support of Mardin Artuklu University) (MAÜ-BAP-14-SHMYO-09).