Materials Science and Engineering C 37 (2014) 278–285
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Graphene oxide based magnetic nanocomposites for efficient treatment of breast cancer Nilesh S. Chaudhari a, Abhijeet P. Pandey a, Pravin O. Patil a, Avinash R. Tekade b, Sanjay B. Bari a, Prashant K. Deshmukh a,⁎ a b
Department of Pharmaceutics, H. R. Patel Institute of Pharmaceutical Education and Research, Karwand Naka, Shirpur. Dist., Dhule 425 405, Maharashtra, India Department of Pharmaceutics, Rajarshi Shahu College of Pharmacy and Research, Tathawade, Pune 411 033, Maharashtra, India
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Article history: Received 21 October 2013 Received in revised form 5 December 2013 Accepted 5 January 2014 Available online 10 January 2014 Keywords: Ferrofluid Breast cancer Anastrozole MCF-7
a b s t r a c t The present work reports a simple one step synthesis of nanoscale graphene oxide magnetic composites (GO–IO) using ferrofluid (GO–IOF). The obtained GO–IO were compared with GO–IO obtained from in situ (GO–IOI) methods. Anastrozole (ANS) was loaded on the GO–IOI and GO–IOF via simple stirring method to form GO– IOA and GO–IOFA respectively. These GO–IO prepared by two techniques were characterized using spectroscopic techniques and vibrating sample magnetometer (VSM) analysis. Particle size and potential were measured using Malvern Zetasizer. Scanning electron microscopy (SEM) was used for studying the surface morphology of GO–IO, and in addition to this elemental analysis was also performed for confirming the presence of iron. The cell viability assay was carried out using the MCF-7 cell line. It revealed that GO–IOFA had reasonably high cytotoxicity (49.7%) compared to GO (13.1%), ANS (16.6), GO–IOI (13%), GO–IOF (13.6) and GO–IOIA (18.34%). Both, GO–IOIA and GO–IOFA showed improved cytotoxicity when compared with pure ANS. GO–IOF were found to exhibit superior magnetic activity due to higher iron content along with smaller particle size and higher loading efficiency compared to GO–IOI. The overall effect suggests that GO–IO can be utilized as efficient carriers for the loading of chemotherapeutic agents. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Development of new drug delivery system has always been an area of interest for formulation developers. Recently, nanomaterials-based drug carriers have made a huge amount of interest owing to their unique properties and significant research is being conducted for interfacing these nanomaterials with a drug delivery system to explore nanotechnology in medicine. The unique properties of these nanomaterials include high and efficient drug loading, targeted delivery and controlled release of drugs and increased stability to name a few [1–5]. Graphene based nanomaterials have recently attracted attention for its use in drug delivery. ‘Graphene’ is the name assigned to a twodimensional sheet of sp2-hybridized carbon. It consists of a singleatom-thick planar sheet and possesses an unusual crystal and electronic tones. The aspect of graphene that is of greatest interest at the present time is its oxidized counterpart, graphene oxide (GO). GO has large p conjugated structure, which can form p–p stacking interaction with aromatic drug. Apart from p–p stacking interaction, presence of abundant functional groups such as carboxyl, hydroxyl, and epoxide functional ⁎ Corresponding author at: Department of Pharmaceutics, H. R. Patel Institute of Pharmaceutical Education and Research, Karwand Naka, Shirpur, Dist., Dhule 425 405, Maharashtra, India. Tel.: +91 9923456365(mobile). E-mail address: [email protected] (P.K. Deshmukh). 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2014.01.007
groups on the edge, top, and bottom surfaces of GO further enhances its interaction with aromatic groups of drug. The interaction of these functional groups to aromatic groups of drug results in the formation of strong hydrogen bond which forms the basis of drug loading on GO and related structures. On the basis of such interaction, researchers reported the loading of various drugs including anticancer drugs on GO for the treatment of tumors, such as doxorubicin, camptothecin, ibuprofen and 5-fluorouracil through non-covalent physisorption (p–p stacking, van der Waals interaction or hydrogen bond) [6–14]. The targeting efficiency of GO based nanocarriers can be enhanced by using GO magnetic composites using iron oxide nanoparticles. Magnetic iron oxide (magnetite Fe3O4 or maghemite gamma-Fe2O3) nanoparticles exhibit super-paramagnetic nature, but suffer the problem of aggregation which limits their magnetic properties and also alters its stability. This problem may be solved by using GO based magnetic composites which will minimize the clumping/aggregation of magnetic nanoparticles thereby increasing the stability as well as preserving their unique magnetic property [15]. Various chemistry based processing methods have been reported for fabrication of graphene/ magnetite nanocomposites (GO–IO) which includes in situ formation [16], solvothermal method [17], hydrothermal method [18], covalent bonding method [19], and electrochemical method [20]. These GO–IO possess unique properties including generation of heat in alternating magnetic fields or an ability to be pointed to a specific tissue or organ
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under influence of external magnetic field. The power generation of heat under alternating magnetic field can be used in cancer therapy. The present investigation explores the different advantages of GO and magnetic nanoparticles for fabrication of an efficient platform for the delivery of anastrozole (ANS). This study demonstrates that GO based GO–IO can be used as efficient nanocarriers for the loading and delivery of water insoluble anticancer drug. In the present study, a novel one step method for the synthesis of GO–IO has been developed. The study involves the comparative study of GO–IO fabricated using in situ (GO–IOI) method and ferrofluid (GO–IOF) conjugation method. Use of ferrofluid for fabricating GO–IO is simple, efficient and less time consuming. To the best of our knowledge, we are the first who utilized ferrofluid for fabricating GO–IO and successfully loaded ANS on prepared GO–IO for treatment of breast cancer.
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were washed with deionized water three times, separated using magnet and finally dried at 60 °C. 2.4. Synthesis of GO–IOF (ferrofluid technique)
2. Experimental
The GO–IO was prepared using ferrofluid (GO–IOF). Initially GO (100 mg) was dispersed in deionized water (10 mL) with stirring. The dispersion was exfoliated by sonicating the dispersion for 3 h, which led to the conversion of dispersion into viscous solution. The viscosity was decreased by the addition of 5 mL of water and dispersion was further sonicated for 1 h for complete exfoliation. After exfoliation of GO dispersion, 0.2 mL of ferrofluid was added to the GO dispersion. The mixture was sonicated for 30 min followed by stirring for 3 h. Finally, the dispersion was centrifuged at 15000 rpm to obtain GO–IOF. It was washed with deionized water and poured in silica crucible for drying at room temperature.
2.1. Materials
2.5. Loading of ANS
Anastrozole (ANS) was gifted by Natco Pharmaceuticals Pvt. Ltd., Mumbai, Maharashtra. Ferrofluid was generously gifted by Ferro-Tech Pvt. Ltd., Bedford, USA. Graphite was kindly provided by Asbury Carbons, New Jersey. Sulfuric acid and hydrochloric acid were purchased from Merck Specialities Pvt. Ltd., Mumbai, and potassium permanganate and trisodium citrate were purchased from Loba Chemie Chemicals Pvt. Ltd, Mumbai. Hydrogen peroxide was purchased from RFCL Limited, Mumbai. Ferric chloride, ferrous sulfate and ammonia hydroxide were purchased from Himedia Lab Pvt. Ltd, Mumbai. All other chemicals and reagents were of analytical grade.
The loading of ANS on synthesized GO–IO was performed using a passive loading method. ANS was dissolved in acetone at a concentration of 10 mg/mL. GO–IOI and GO–IOF were dispersed in deionized water and sonicated for 30 min, separately. After sonication, both GO–IO dispersions were mixed into drug solution with slow stirring at room temperature for 24 h. The drug solution was then centrifuged at 16000 rpm for 30 min to obtain ANS loaded GO–IO i.e. GO–IOIA and GO–IOFA. Obtained GO–IO pellets were washed three times with deionized water. The concentration of ANS in the supernatant was measured using standard curve of ANS. The loading efficiency of GO–IO was calculated using the following formula
2.2. Synthesis of GO GO was synthesized from graphite flakes by an oxidation process using modified Hummers method [21,22]. Graphite flakes (2 g) were dissolved in 50 mL of conc. H2SO4 by maintaining the temperature at 0 °C with vigorously stirring for 3 h. To this solution, KMnO4 (6 g) was added slowly while keeping the temperature below 15 °C. The KMnO4 was added slowly for 2 h, after which the mixture was kept for stirring for further 1 h. The temperature was slowly raised to 35 °C, keeping the mixture under stirring until the mixture becomes pasty brownish; which was accompanied by an increase in viscosity of the mixture. The pasty brownish mixture was diluted with deionized water, followed by the slow addition of H2O2 (7 mL) to arrest the oxidation process which alters the color of mixture from dark brown to bright yellow, indicating the high level of oxidation of graphite. The obtained mixture was centrifuged at 25 000 rpm to collect the pellets of GO. Recovery of settled pellets was performed by simple decantation of supernatant. The obtained GO was washed three times with an HCl aqueous solution (1 M) followed by repeated rinsing with deionized water until the pH of GO dispersion reached 4–5 and dried at 35 °C. 2.3. Synthesis of GO–IOI (in situ synthesis) The in situ synthesis of GO–IO (GO–IOI) was performed according to previously described method with slight modification [23]. Initially, GO (1.5 g) was dispersed in deionized water (100 mL) by stirring. The dispersion was exfoliated by sonication to obtain evenly distributed GO sheets. The exfoliation leads to thickening of GO dispersion which confirms the exfoliation of GO. The exfoliated GO dispersion was mixed in three necked flask followed by addition of a mixture of FeCl3 (0.33 g) and FeSO4 (0.38 g) in 100 mL deionized water. The resultant mixture was heated up to 80 °C with continuous stirring and finally the pH of the mixture was adjusted to 12 with the addition of 25% ammonia solution. After addition of ammonia solution, tri-sodium citrate (1 g) was added to the mixture and kept at 95 °C for 1 h. The resultant black color suspension was centrifuged at 15 000 rpm. The obtained GO–IOI
Loading efficiency ð%LEÞ ¼
ðDtotal−DsupernatantÞ 100: Dtotal
The drug loading ability of GO–IO was calculated using the following formula Drug loading ability ð%DLÞ ¼
DGO–IO 100: WGO–IO
Where, Dtotal = total amount of ANS taken; Dsupernatant = amount of ANS present in supernatant; DGO–IO = amount of ANS on loaded GO–IO; WGO–IO = total amount of GO–IO taken. 2.6. Characterization 2.6.1. Spectroscopic analysis Ultraviolet–visible (UV–vis) absorption spectra of plain GO, IO, GO–IOI and GO–IOF were measured with a spectrophotometer (UV-1800 PC, Shimadzu, Japan), where the light path length was 1 cm. FTIR spectra of samples were recorded on IR spectrometer (Fourier Transform Infrared Spectroscopy–IR Affinity 1700, Shimadzu, Japan) at a resolution of 4 cm−1 with a maximum of 100 scans at frequencies ranging from 400 to 4000 cm−1. For getting the IR spectra, the samples were thoroughly mixed with KBr in a ratio of 1:100 in the crucible. The mixture was then filled in powder sampling cells for spectral analysis. 2.6.2. Particle size and zeta potential Particle size analysis of GO, GO–IOI and GO–IOF was carried out using a Zetasizer (Nano ZS 90, Malvern Instruments Ltd., Malvern, UK) equipped with a 4.0 mW internal laser, which works on the principle of dynamic light scattering. The samples were diluted with doubledistilled water in a disposable polystyrene cell prior to measurements to obtain dispersion with a concentration below 0.5 mg/mL (to avoid multiple scattering). All measurements were performed at 25 °C, at a
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scattering angle of 90°. The intensity-weighed mean diameter of the bulk population of particles was given by the z-average diameter value of the particles. Each sample was analyzed in triplicate. The analysis of zeta potential was done using a Malvern ZS 90 Zetasizer and a folded capillary cell. Before the sample was used for zeta potential measurement, it was processed similar to that used in the particle size analysis study. The zeta potential measurement was carried out subsequently. All tests were conducted at 25 °C in triplicate. 2.6.3. Study of surface morphology and elemental analysis The surface morphology of GO, ferrofluid, GO–IOI and GO–IOF was studied using scanning electron microscopy (SEM). SEM analysis was done by keeping the samples on aluminum stub and gold coating prior to analysis using Philips LEO 1530-2 FESEM/EDS, with 20 kV accelerating voltages. The surface morphology of all the samples was compared. Elemental analysis of GO, GO–IOI and GO–IOF was done using energy dispersive spectroscopy using Philips LEO 1530-2 FESEM/EDS, with 15 kV accelerating voltages. 2.6.4. Magnetic susceptibility Preliminary magnetic susceptibility of GO–IOI and GO–IOF was studied using an external magnet. The magnetic properties of GO–IOI and GO–IOF were also studied by measuring the magnetic susceptibility as a function of the applied magnetic field H using a vibrating sample magnetometer (Lakeshore VSM 7410) with a maximum applied magnetic field of 20 kOe. The hysteresis of the magnetization was obtained by varying H between +20000 and −20 000 Oe at 300 K. 2.6.5. In vitro cell viability assay In vitro anticancer activity of GO, ANS, GO–IOI, GO–IOF, GO–IOIA and GO–IOFA was studied with regard to cell viability of MCF-7 breast cancer cell line using MTT assay. The MCF-7 breast cancer cells were grown in a 96-well plate and left for seeding for 24 h. After 24 h seeding, the old medium was discarded and the cells were incubated with various concentrations of GO, ANS, GO–IOI, GO–IOF, GO–IOIA and GO–IOFA (0, 12, 20, 40, 80 μg/mL). The cells were incubated for further 24 h at 37 °C with 5% CO2. The medium was replaced with serum-free DMEM before addition of 20 μL MTT (5 mg/mL) in PBS. After 4 h, when the MTT fully integrated with cells, 150 μL DMSO was added to the cells. The plates were then oscillated for 10 min to confirm the dissolution of Formazan in DMSO. Finally, the absorbance at 570 nm was measured by using a TRITURUS microplate reader. 3. Results and discussion 3.1. Spectroscopic analysis The UV–vis spectrum of GO (A), IO (B), GO–IOI (C) and GO–IOF (D) is shown in Fig. 1. The UV absorption of GO and IO appeared at 230 nm as a small peak and 280 nm as a small broad peak respectively, whereas the UV absorbance of the final product GO–IOI and GO–IOF appeared at approximately 230 nm as a very broad hump along with a second broad peak at around 260 nm which was absent in UV spectrum of pure GO. GO dispersed in water exhibits a maximum absorption at 230 nm, attributed to the p–p* transition resulting from C_C bonds of the aromatic skeleton. The appearance of a broad peak at 260 nm along with a small broad peak between 380 and 390 nm can be attributed to the presence of iron nanoparticle conjugated to oxygen containing functional groups present on GO [24,25]. The difference between the UV spectra of GO (A), IO (B), GO–IOI (C) and GO–IOF (D) confirms the change in GO absorbance, which can be correlated to the conjugation of iron nanoparticles on GO structure. No significant change was observed between the absorbance curves of GO–IOI (C) and GO–IOF (D) which suggests that both methods are capable of binding iron oxide nanoparticles on GO structure. The mechanism of the conjugation of IO with GO has not been well defined to date. Recently, it has been
Fig. 1. UV–vis spectra of plain GO (A), IO (B), GO–IOI (C) and GO–IOF (D).
reported that the conjugation of IO with GO proceeds with hydrolysis of IO nanoparticles during incubation leading to release of hydroxyl ions slowly and uniformly resulting in the formation of Fe(OH)3. The Fe(OH)3 particles produced after hydrolysis then get attached onto the surface of the GO through multiple oxygen containing functional groups, such as carboxyl, hydroxyl, and epoxyl [26]. The formation of GO–IO was also confirmed by FTIR spectroscopy (Fig. 2). In the IR spectrum of GO, the peak at 1761 cm−1 corresponded to the stretching band of C\O in carboxylic acid or carbonyl moieties. The intense bands at 3128 and 1226 cm−1 were attributed to stretching and bending of the O\H, respectively [12]. The deformation of the C\O was observed at 1058 cm−1. In the FTIR spectrum of GO–IOI (B), the difference was evidenced by the shifting of the peaks of C\O and O\H at 1726 cm− 1 and 3130 cm−1, respectively. The peak was observed at around 580 cm− 1 (peak not marked) which can be attributed to Fe\O [27]. In the case of GO–IOF, the peaks were similar to that of GO–IOI except that the peak near 580 cm−1 (peak not marked) was more intense, which suggests the superiority of ferrofluid technique over in situ method for the preparation of GO–IO. In the IR spectra of ANS loaded GO–IO, the peaks at 2947 cm− 1, 2234 cm−1 (peak not marked), and 1585 cm−1 show the presence of aromatic rings, C`N stretch and C_N stretch respectively which confirms the conjugation of ANS on GO–IO. 3.2. Particle size and zeta potential Analysis of particle size and zeta potential was done by observing the change in size and potential of plain GO, GO–IOI and GO–IOF (Fig. 3). The particle size of iron oxide nanoparticles present in ferrofluid was 10 nm and had the negative potential of −23.4 mV. The plain GO had a size of 153 nm and a negative potential of −16.3 mV. In the case of
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Fig. 2. FT-IR spectra of GO (A), GO–IOI (B), GO–IOF (C) and GO–IOFA (D).
GO–IOI and GO–IOF, a shift in the value of zeta potential to more negative values was observed which can be attributed to conjugation of iron oxide nanoparticles on the structure of GO. A similar event was observed in results of particle size, which is very obvious due to binding of iron oxide nanoparticles on GO. The negative potential of GO–IOI prepared (− 26.0 mV) was more significant as compared to GO–IOF (− 22.3 mV). This variation may be correlated to the fact that
surfactants are employed in the case of ferrofluid to stabilize iron oxide nanoparticles and minimize their aggregation. So, the surfactant might lead to decrease in negative potential of iron oxide nanoparticles which ultimately leads to decrease in zeta potential of GO–IO. The variation in particle size was more prominent for GI–IOI (477.7 nm) as compared to GO–IOF (247.5 nm). This variation may be correlated to the fact that the iron oxide nanoparticles in ferrofluid were very less
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which is not the case with the GO. Apart from the stacked sheet structure of GO, the other difference of pristine graphene from GO is the absence of oxygen containing functional groups. The EDX analysis showed the presence of oxygen which is attributed to oxygen containing functional groups present on GO. EDX elemental measurements enabled us to see the elemental contents of GO–IO. The EDX analysis was carried out for GO, GO–IOF and GO–IOI as a support for the FT-IR results (Fig. 5). The results of elemental analysis show the presence of Fe in both GO–IOI and GO–IOF. In the case of GO, peaks for carbon (C) and oxygen (O) were detected. An additional single peak of Fe was observed for GO– IOI and two Fe peaks were observed for GO–IOF, which suggests that the GO–IOF contain more amount of iron as compared to GO–IOI. Fig. 3. Particle size (bar) and zeta potential (line) of ferrofluid, GO, GO–IOI and GO–IOF.
3.4. Magnetic susceptibility measurements as compared to the particle size of iron oxide as obtained via in situ method. The result indicates that the ferrofluid method is superior to in situ method with respect to particle size for the preparation of GO–IO. 3.3. Study of surface morphology and elemental analysis Surface morphology was analyzed using SEM analysis. The SEM images of plain GO, ferrofluid, GO–IOI and GO–IOF are shown in Fig. 4. The GO can be observed as the stacked structure of GO nanosheets along with spherical shaped iron oxide nanoparticles of ferrofluid. In the case of GO–IOF, the stacked structure of GO nanosheets is converted to a smooth structure with freckles. In the case of GO–IOI, the structure is rough as compared to GO–IOF which can be attributed to the larger diameter of iron oxide nanoparticles deposited on GO structure. The SEM images clearly indicate the change in surface morphology which suggests the conjugation of iron oxide nanoparticles with GO surface which is also supported by the results of spectroscopic analysis along with results of particle size and potential analysis. The SEM image of pristine graphene is also rough, similar to GO but it does not show the presence of multiple stacked sheets which is visible in the SEM image of GO and features a curly morphology with a thin, wrinkled structure
The superparamagnetic behavior of GO–IOF (A, C) and GO–IOI (B, D) was verified using an external magnet as shown in Fig. 6. The figure shows the aggregation of GO–IO on the side walls and top of the tube. The figure suggests that the formed GO–IOF and GO–IOI move under the influence of external magnetic field which further confirms the formation of magnetic GO–IO. Apart from a preliminary study, the magnetic properties of the resulting GO–IO at room temperature were characterized by a vibrating sample magnetometer (VSM). The obtained hysteresis loops were S shaped (Fig. 7). Although the GO–IOF (B) hybrid exhibits a superparamagnetic state with low remnant magnetization at room temperature as compared to GO–IOI (A), the magnetizations of both GO–IO hybrids reach saturation and their magnetic properties were found to be good. The superior magnetic property of GO–IOF in comparison to GO–IOI can also be correlated to the iron content in both GO–IO, as evident from the results of elemental analysis. The percentage of iron bound to GO was higher in the case of GO–IOF as compared to GO–IOI which may be correlated to the fact that the particle size of an iron nanoparticle present in ferrofluid was much smaller as compared to iron nanoparticles formed in GO–IOI. The smaller size of nanoparticles provides space for the inclusion of the higher percentage
Fig. 4. Surface morphology of GO (A), GO–IOI (B), GO–IOF (C) and ferrofluid (D).
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Fig. 5. a.Elemental analysis of GO. b. Elemental analysis of GO–IOF (A) and GO–IOI (B).
of iron as the void space present between iron nanoparticles bound on the GO is minimized. 3.4.1. In vitro cell viability assay Cell viability was assessed using MTT assay. The result is shown in Fig. 8. The results of the present study demonstrated that dispersion of ANS loaded GO–IO resulted in a decrease in cell viability as compared to ANS. Pure ANS and GO showed 16.6% and 13.1% inhibition of cell viability respectively. Between GO–IO and GO–IOF, GO–IOF showed higher inhibition of cell viability as compared to GO–IOI which may be correlated to the small size of GO–IOF in comparison to GO–IOI. Another aspect of GO–IOF which can be correlated to its higher anti-proliferative activity is the number of IO nanoparticles conjugated on the GO. Due to the smaller size of IO nanoparticles present in a ferrofluid, higher number of IO nanoparticles could have been conjugated to GO. Apart from enhanced penetration due to smaller size, another aspect of IO which contributes to the enhanced anti-proliferative activity of GO–IO and GO–IOF as compared to ANS is the inherent anti-proliferative activity
of IO itself. According to recently published literature, it has been demonstrated that the magnetic IO nanoparticles show good antiproliferative activity against breast cancer MCF-7 cells [28]. Although the present results showed good antiproliferative activity of magnetic nanocomposites as compared to pure drug, the concentration of magnetic nanocomposites required can still be decreased. The particle size can be further decreased along with an increase in net positive charge for enhancing the anti-proliferative activity of the fabricated magnetic nanocomposites using the minimum quantity of drug loaded magnetic nanocomposites which will be much better from a patient's point of view. The loading of drug can also be increased for decreasing net concentration of magnetic nanocomposites. The overall result suggests that the inhibition of cell viability was dose dependent. The present findings give a clear outline about the superiority of GO magnetic nanocomposites for treatment of breast cancer as compared to plain ANS. The local hyperthermia caused by IO can play a vital role in enhancing its antiproliferative activity which in turns depends on the magnetic susceptibility, size and density of IO near tumor cells. From the study, we can
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Fig. 6. Magnetic activity of GO–IOF (A—side view, C—aerial view) and GO–IOI (B—side view, D—aerial view).
Fig. 7. Magnetic hysteresis loop of GO–IOI (A) and GO–IOF (B).
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Fig. 8. Cell viability assay of GO, ANS, GO–IOI, GO–IOF, GO–IOFA, GO–IOIA and ADR.
conclude that the loading of ANS on GO–IO enhances the anticancer activity, which means that with increasing solubility and stability in physiological solutions, the GO nanocarriers could indirectly enhance the dissolution of water insoluble anticancer drugs and so is the case with anticancer activity.
4. Conclusions In the present work, we have reported the synthesis of graphene based GO–IO, using two different methods and ferrofluid method was found to be superior with respect to iron content along with magnetic property of prepared GO–IO. Both, the GO–IOIA (74.3%) and GO–IOFA (84%) showed high loading efficiency. Particle size of GO–IOF was lesser than GO–IOI which may have effects on cellular uptake and ultimately anticancer activity. This fact has been confirmed by the results of cell viability assay in which GO–IOFA exhibited much higher cytotoxicity to MCF-7 cells than GO–IOIA and pure ANS. Taken together, this study demonstrates the viability of utilizing GO–IO as nanocarriers for targeted delivery of drugs, which may have potential clinical advantages pertaining to increased therapeutic efficacy and decreased local toxicity.
Acknowledgments The authors gratefully acknowledge Asbury Carbons, New Jersey for generously gifting graphite. We gratefully acknowledge Ferro-Tech Pvt. Ltd., USA for providing the gift samples of ferrofluid. We are also thankful to ACTREC, Mumbai (India) and SAIF, Indian Institute of Technology, Madras for carrying out in-vitro cell viability assay and VSM analysis, respectively.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Graphene oxide based magnetic nanocomposites for efficient treatment of breast cancer Nilesh S. Chaudhari a, Abhijeet P. Pandey a, Pravin O. Patil a, Avinash R. Tekade b, Sanjay B. Bari a, Prashant K. Deshmukh a,⁎ a b
Department of Pharmaceutics, H. R. Patel Institute of Pharmaceutical Education and Research, Karwand Naka, Shirpur. Dist., Dhule 425 405, Maharashtra, India Department of Pharmaceutics, Rajarshi Shahu College of Pharmacy and Research, Tathawade, Pune 411 033, Maharashtra, India
a r t i c l e
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Article history: Received 21 October 2013 Received in revised form 5 December 2013 Accepted 5 January 2014 Available online 10 January 2014 Keywords: Ferrofluid Breast cancer Anastrozole MCF-7
a b s t r a c t The present work reports a simple one step synthesis of nanoscale graphene oxide magnetic composites (GO–IO) using ferrofluid (GO–IOF). The obtained GO–IO were compared with GO–IO obtained from in situ (GO–IOI) methods. Anastrozole (ANS) was loaded on the GO–IOI and GO–IOF via simple stirring method to form GO– IOA and GO–IOFA respectively. These GO–IO prepared by two techniques were characterized using spectroscopic techniques and vibrating sample magnetometer (VSM) analysis. Particle size and potential were measured using Malvern Zetasizer. Scanning electron microscopy (SEM) was used for studying the surface morphology of GO–IO, and in addition to this elemental analysis was also performed for confirming the presence of iron. The cell viability assay was carried out using the MCF-7 cell line. It revealed that GO–IOFA had reasonably high cytotoxicity (49.7%) compared to GO (13.1%), ANS (16.6), GO–IOI (13%), GO–IOF (13.6) and GO–IOIA (18.34%). Both, GO–IOIA and GO–IOFA showed improved cytotoxicity when compared with pure ANS. GO–IOF were found to exhibit superior magnetic activity due to higher iron content along with smaller particle size and higher loading efficiency compared to GO–IOI. The overall effect suggests that GO–IO can be utilized as efficient carriers for the loading of chemotherapeutic agents. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Development of new drug delivery system has always been an area of interest for formulation developers. Recently, nanomaterials-based drug carriers have made a huge amount of interest owing to their unique properties and significant research is being conducted for interfacing these nanomaterials with a drug delivery system to explore nanotechnology in medicine. The unique properties of these nanomaterials include high and efficient drug loading, targeted delivery and controlled release of drugs and increased stability to name a few [1–5]. Graphene based nanomaterials have recently attracted attention for its use in drug delivery. ‘Graphene’ is the name assigned to a twodimensional sheet of sp2-hybridized carbon. It consists of a singleatom-thick planar sheet and possesses an unusual crystal and electronic tones. The aspect of graphene that is of greatest interest at the present time is its oxidized counterpart, graphene oxide (GO). GO has large p conjugated structure, which can form p–p stacking interaction with aromatic drug. Apart from p–p stacking interaction, presence of abundant functional groups such as carboxyl, hydroxyl, and epoxide functional ⁎ Corresponding author at: Department of Pharmaceutics, H. R. Patel Institute of Pharmaceutical Education and Research, Karwand Naka, Shirpur, Dist., Dhule 425 405, Maharashtra, India. Tel.: +91 9923456365(mobile). E-mail address: [email protected] (P.K. Deshmukh). 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2014.01.007
groups on the edge, top, and bottom surfaces of GO further enhances its interaction with aromatic groups of drug. The interaction of these functional groups to aromatic groups of drug results in the formation of strong hydrogen bond which forms the basis of drug loading on GO and related structures. On the basis of such interaction, researchers reported the loading of various drugs including anticancer drugs on GO for the treatment of tumors, such as doxorubicin, camptothecin, ibuprofen and 5-fluorouracil through non-covalent physisorption (p–p stacking, van der Waals interaction or hydrogen bond) [6–14]. The targeting efficiency of GO based nanocarriers can be enhanced by using GO magnetic composites using iron oxide nanoparticles. Magnetic iron oxide (magnetite Fe3O4 or maghemite gamma-Fe2O3) nanoparticles exhibit super-paramagnetic nature, but suffer the problem of aggregation which limits their magnetic properties and also alters its stability. This problem may be solved by using GO based magnetic composites which will minimize the clumping/aggregation of magnetic nanoparticles thereby increasing the stability as well as preserving their unique magnetic property [15]. Various chemistry based processing methods have been reported for fabrication of graphene/ magnetite nanocomposites (GO–IO) which includes in situ formation [16], solvothermal method [17], hydrothermal method [18], covalent bonding method [19], and electrochemical method [20]. These GO–IO possess unique properties including generation of heat in alternating magnetic fields or an ability to be pointed to a specific tissue or organ
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under influence of external magnetic field. The power generation of heat under alternating magnetic field can be used in cancer therapy. The present investigation explores the different advantages of GO and magnetic nanoparticles for fabrication of an efficient platform for the delivery of anastrozole (ANS). This study demonstrates that GO based GO–IO can be used as efficient nanocarriers for the loading and delivery of water insoluble anticancer drug. In the present study, a novel one step method for the synthesis of GO–IO has been developed. The study involves the comparative study of GO–IO fabricated using in situ (GO–IOI) method and ferrofluid (GO–IOF) conjugation method. Use of ferrofluid for fabricating GO–IO is simple, efficient and less time consuming. To the best of our knowledge, we are the first who utilized ferrofluid for fabricating GO–IO and successfully loaded ANS on prepared GO–IO for treatment of breast cancer.
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were washed with deionized water three times, separated using magnet and finally dried at 60 °C. 2.4. Synthesis of GO–IOF (ferrofluid technique)
2. Experimental
The GO–IO was prepared using ferrofluid (GO–IOF). Initially GO (100 mg) was dispersed in deionized water (10 mL) with stirring. The dispersion was exfoliated by sonicating the dispersion for 3 h, which led to the conversion of dispersion into viscous solution. The viscosity was decreased by the addition of 5 mL of water and dispersion was further sonicated for 1 h for complete exfoliation. After exfoliation of GO dispersion, 0.2 mL of ferrofluid was added to the GO dispersion. The mixture was sonicated for 30 min followed by stirring for 3 h. Finally, the dispersion was centrifuged at 15000 rpm to obtain GO–IOF. It was washed with deionized water and poured in silica crucible for drying at room temperature.
2.1. Materials
2.5. Loading of ANS
Anastrozole (ANS) was gifted by Natco Pharmaceuticals Pvt. Ltd., Mumbai, Maharashtra. Ferrofluid was generously gifted by Ferro-Tech Pvt. Ltd., Bedford, USA. Graphite was kindly provided by Asbury Carbons, New Jersey. Sulfuric acid and hydrochloric acid were purchased from Merck Specialities Pvt. Ltd., Mumbai, and potassium permanganate and trisodium citrate were purchased from Loba Chemie Chemicals Pvt. Ltd, Mumbai. Hydrogen peroxide was purchased from RFCL Limited, Mumbai. Ferric chloride, ferrous sulfate and ammonia hydroxide were purchased from Himedia Lab Pvt. Ltd, Mumbai. All other chemicals and reagents were of analytical grade.
The loading of ANS on synthesized GO–IO was performed using a passive loading method. ANS was dissolved in acetone at a concentration of 10 mg/mL. GO–IOI and GO–IOF were dispersed in deionized water and sonicated for 30 min, separately. After sonication, both GO–IO dispersions were mixed into drug solution with slow stirring at room temperature for 24 h. The drug solution was then centrifuged at 16000 rpm for 30 min to obtain ANS loaded GO–IO i.e. GO–IOIA and GO–IOFA. Obtained GO–IO pellets were washed three times with deionized water. The concentration of ANS in the supernatant was measured using standard curve of ANS. The loading efficiency of GO–IO was calculated using the following formula
2.2. Synthesis of GO GO was synthesized from graphite flakes by an oxidation process using modified Hummers method [21,22]. Graphite flakes (2 g) were dissolved in 50 mL of conc. H2SO4 by maintaining the temperature at 0 °C with vigorously stirring for 3 h. To this solution, KMnO4 (6 g) was added slowly while keeping the temperature below 15 °C. The KMnO4 was added slowly for 2 h, after which the mixture was kept for stirring for further 1 h. The temperature was slowly raised to 35 °C, keeping the mixture under stirring until the mixture becomes pasty brownish; which was accompanied by an increase in viscosity of the mixture. The pasty brownish mixture was diluted with deionized water, followed by the slow addition of H2O2 (7 mL) to arrest the oxidation process which alters the color of mixture from dark brown to bright yellow, indicating the high level of oxidation of graphite. The obtained mixture was centrifuged at 25 000 rpm to collect the pellets of GO. Recovery of settled pellets was performed by simple decantation of supernatant. The obtained GO was washed three times with an HCl aqueous solution (1 M) followed by repeated rinsing with deionized water until the pH of GO dispersion reached 4–5 and dried at 35 °C. 2.3. Synthesis of GO–IOI (in situ synthesis) The in situ synthesis of GO–IO (GO–IOI) was performed according to previously described method with slight modification [23]. Initially, GO (1.5 g) was dispersed in deionized water (100 mL) by stirring. The dispersion was exfoliated by sonication to obtain evenly distributed GO sheets. The exfoliation leads to thickening of GO dispersion which confirms the exfoliation of GO. The exfoliated GO dispersion was mixed in three necked flask followed by addition of a mixture of FeCl3 (0.33 g) and FeSO4 (0.38 g) in 100 mL deionized water. The resultant mixture was heated up to 80 °C with continuous stirring and finally the pH of the mixture was adjusted to 12 with the addition of 25% ammonia solution. After addition of ammonia solution, tri-sodium citrate (1 g) was added to the mixture and kept at 95 °C for 1 h. The resultant black color suspension was centrifuged at 15 000 rpm. The obtained GO–IOI
Loading efficiency ð%LEÞ ¼
ðDtotal−DsupernatantÞ 100: Dtotal
The drug loading ability of GO–IO was calculated using the following formula Drug loading ability ð%DLÞ ¼
DGO–IO 100: WGO–IO
Where, Dtotal = total amount of ANS taken; Dsupernatant = amount of ANS present in supernatant; DGO–IO = amount of ANS on loaded GO–IO; WGO–IO = total amount of GO–IO taken. 2.6. Characterization 2.6.1. Spectroscopic analysis Ultraviolet–visible (UV–vis) absorption spectra of plain GO, IO, GO–IOI and GO–IOF were measured with a spectrophotometer (UV-1800 PC, Shimadzu, Japan), where the light path length was 1 cm. FTIR spectra of samples were recorded on IR spectrometer (Fourier Transform Infrared Spectroscopy–IR Affinity 1700, Shimadzu, Japan) at a resolution of 4 cm−1 with a maximum of 100 scans at frequencies ranging from 400 to 4000 cm−1. For getting the IR spectra, the samples were thoroughly mixed with KBr in a ratio of 1:100 in the crucible. The mixture was then filled in powder sampling cells for spectral analysis. 2.6.2. Particle size and zeta potential Particle size analysis of GO, GO–IOI and GO–IOF was carried out using a Zetasizer (Nano ZS 90, Malvern Instruments Ltd., Malvern, UK) equipped with a 4.0 mW internal laser, which works on the principle of dynamic light scattering. The samples were diluted with doubledistilled water in a disposable polystyrene cell prior to measurements to obtain dispersion with a concentration below 0.5 mg/mL (to avoid multiple scattering). All measurements were performed at 25 °C, at a
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scattering angle of 90°. The intensity-weighed mean diameter of the bulk population of particles was given by the z-average diameter value of the particles. Each sample was analyzed in triplicate. The analysis of zeta potential was done using a Malvern ZS 90 Zetasizer and a folded capillary cell. Before the sample was used for zeta potential measurement, it was processed similar to that used in the particle size analysis study. The zeta potential measurement was carried out subsequently. All tests were conducted at 25 °C in triplicate. 2.6.3. Study of surface morphology and elemental analysis The surface morphology of GO, ferrofluid, GO–IOI and GO–IOF was studied using scanning electron microscopy (SEM). SEM analysis was done by keeping the samples on aluminum stub and gold coating prior to analysis using Philips LEO 1530-2 FESEM/EDS, with 20 kV accelerating voltages. The surface morphology of all the samples was compared. Elemental analysis of GO, GO–IOI and GO–IOF was done using energy dispersive spectroscopy using Philips LEO 1530-2 FESEM/EDS, with 15 kV accelerating voltages. 2.6.4. Magnetic susceptibility Preliminary magnetic susceptibility of GO–IOI and GO–IOF was studied using an external magnet. The magnetic properties of GO–IOI and GO–IOF were also studied by measuring the magnetic susceptibility as a function of the applied magnetic field H using a vibrating sample magnetometer (Lakeshore VSM 7410) with a maximum applied magnetic field of 20 kOe. The hysteresis of the magnetization was obtained by varying H between +20000 and −20 000 Oe at 300 K. 2.6.5. In vitro cell viability assay In vitro anticancer activity of GO, ANS, GO–IOI, GO–IOF, GO–IOIA and GO–IOFA was studied with regard to cell viability of MCF-7 breast cancer cell line using MTT assay. The MCF-7 breast cancer cells were grown in a 96-well plate and left for seeding for 24 h. After 24 h seeding, the old medium was discarded and the cells were incubated with various concentrations of GO, ANS, GO–IOI, GO–IOF, GO–IOIA and GO–IOFA (0, 12, 20, 40, 80 μg/mL). The cells were incubated for further 24 h at 37 °C with 5% CO2. The medium was replaced with serum-free DMEM before addition of 20 μL MTT (5 mg/mL) in PBS. After 4 h, when the MTT fully integrated with cells, 150 μL DMSO was added to the cells. The plates were then oscillated for 10 min to confirm the dissolution of Formazan in DMSO. Finally, the absorbance at 570 nm was measured by using a TRITURUS microplate reader. 3. Results and discussion 3.1. Spectroscopic analysis The UV–vis spectrum of GO (A), IO (B), GO–IOI (C) and GO–IOF (D) is shown in Fig. 1. The UV absorption of GO and IO appeared at 230 nm as a small peak and 280 nm as a small broad peak respectively, whereas the UV absorbance of the final product GO–IOI and GO–IOF appeared at approximately 230 nm as a very broad hump along with a second broad peak at around 260 nm which was absent in UV spectrum of pure GO. GO dispersed in water exhibits a maximum absorption at 230 nm, attributed to the p–p* transition resulting from C_C bonds of the aromatic skeleton. The appearance of a broad peak at 260 nm along with a small broad peak between 380 and 390 nm can be attributed to the presence of iron nanoparticle conjugated to oxygen containing functional groups present on GO [24,25]. The difference between the UV spectra of GO (A), IO (B), GO–IOI (C) and GO–IOF (D) confirms the change in GO absorbance, which can be correlated to the conjugation of iron nanoparticles on GO structure. No significant change was observed between the absorbance curves of GO–IOI (C) and GO–IOF (D) which suggests that both methods are capable of binding iron oxide nanoparticles on GO structure. The mechanism of the conjugation of IO with GO has not been well defined to date. Recently, it has been
Fig. 1. UV–vis spectra of plain GO (A), IO (B), GO–IOI (C) and GO–IOF (D).
reported that the conjugation of IO with GO proceeds with hydrolysis of IO nanoparticles during incubation leading to release of hydroxyl ions slowly and uniformly resulting in the formation of Fe(OH)3. The Fe(OH)3 particles produced after hydrolysis then get attached onto the surface of the GO through multiple oxygen containing functional groups, such as carboxyl, hydroxyl, and epoxyl [26]. The formation of GO–IO was also confirmed by FTIR spectroscopy (Fig. 2). In the IR spectrum of GO, the peak at 1761 cm−1 corresponded to the stretching band of C\O in carboxylic acid or carbonyl moieties. The intense bands at 3128 and 1226 cm−1 were attributed to stretching and bending of the O\H, respectively [12]. The deformation of the C\O was observed at 1058 cm−1. In the FTIR spectrum of GO–IOI (B), the difference was evidenced by the shifting of the peaks of C\O and O\H at 1726 cm− 1 and 3130 cm−1, respectively. The peak was observed at around 580 cm− 1 (peak not marked) which can be attributed to Fe\O [27]. In the case of GO–IOF, the peaks were similar to that of GO–IOI except that the peak near 580 cm−1 (peak not marked) was more intense, which suggests the superiority of ferrofluid technique over in situ method for the preparation of GO–IO. In the IR spectra of ANS loaded GO–IO, the peaks at 2947 cm− 1, 2234 cm−1 (peak not marked), and 1585 cm−1 show the presence of aromatic rings, C`N stretch and C_N stretch respectively which confirms the conjugation of ANS on GO–IO. 3.2. Particle size and zeta potential Analysis of particle size and zeta potential was done by observing the change in size and potential of plain GO, GO–IOI and GO–IOF (Fig. 3). The particle size of iron oxide nanoparticles present in ferrofluid was 10 nm and had the negative potential of −23.4 mV. The plain GO had a size of 153 nm and a negative potential of −16.3 mV. In the case of
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Fig. 2. FT-IR spectra of GO (A), GO–IOI (B), GO–IOF (C) and GO–IOFA (D).
GO–IOI and GO–IOF, a shift in the value of zeta potential to more negative values was observed which can be attributed to conjugation of iron oxide nanoparticles on the structure of GO. A similar event was observed in results of particle size, which is very obvious due to binding of iron oxide nanoparticles on GO. The negative potential of GO–IOI prepared (− 26.0 mV) was more significant as compared to GO–IOF (− 22.3 mV). This variation may be correlated to the fact that
surfactants are employed in the case of ferrofluid to stabilize iron oxide nanoparticles and minimize their aggregation. So, the surfactant might lead to decrease in negative potential of iron oxide nanoparticles which ultimately leads to decrease in zeta potential of GO–IO. The variation in particle size was more prominent for GI–IOI (477.7 nm) as compared to GO–IOF (247.5 nm). This variation may be correlated to the fact that the iron oxide nanoparticles in ferrofluid were very less
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which is not the case with the GO. Apart from the stacked sheet structure of GO, the other difference of pristine graphene from GO is the absence of oxygen containing functional groups. The EDX analysis showed the presence of oxygen which is attributed to oxygen containing functional groups present on GO. EDX elemental measurements enabled us to see the elemental contents of GO–IO. The EDX analysis was carried out for GO, GO–IOF and GO–IOI as a support for the FT-IR results (Fig. 5). The results of elemental analysis show the presence of Fe in both GO–IOI and GO–IOF. In the case of GO, peaks for carbon (C) and oxygen (O) were detected. An additional single peak of Fe was observed for GO– IOI and two Fe peaks were observed for GO–IOF, which suggests that the GO–IOF contain more amount of iron as compared to GO–IOI. Fig. 3. Particle size (bar) and zeta potential (line) of ferrofluid, GO, GO–IOI and GO–IOF.
3.4. Magnetic susceptibility measurements as compared to the particle size of iron oxide as obtained via in situ method. The result indicates that the ferrofluid method is superior to in situ method with respect to particle size for the preparation of GO–IO. 3.3. Study of surface morphology and elemental analysis Surface morphology was analyzed using SEM analysis. The SEM images of plain GO, ferrofluid, GO–IOI and GO–IOF are shown in Fig. 4. The GO can be observed as the stacked structure of GO nanosheets along with spherical shaped iron oxide nanoparticles of ferrofluid. In the case of GO–IOF, the stacked structure of GO nanosheets is converted to a smooth structure with freckles. In the case of GO–IOI, the structure is rough as compared to GO–IOF which can be attributed to the larger diameter of iron oxide nanoparticles deposited on GO structure. The SEM images clearly indicate the change in surface morphology which suggests the conjugation of iron oxide nanoparticles with GO surface which is also supported by the results of spectroscopic analysis along with results of particle size and potential analysis. The SEM image of pristine graphene is also rough, similar to GO but it does not show the presence of multiple stacked sheets which is visible in the SEM image of GO and features a curly morphology with a thin, wrinkled structure
The superparamagnetic behavior of GO–IOF (A, C) and GO–IOI (B, D) was verified using an external magnet as shown in Fig. 6. The figure shows the aggregation of GO–IO on the side walls and top of the tube. The figure suggests that the formed GO–IOF and GO–IOI move under the influence of external magnetic field which further confirms the formation of magnetic GO–IO. Apart from a preliminary study, the magnetic properties of the resulting GO–IO at room temperature were characterized by a vibrating sample magnetometer (VSM). The obtained hysteresis loops were S shaped (Fig. 7). Although the GO–IOF (B) hybrid exhibits a superparamagnetic state with low remnant magnetization at room temperature as compared to GO–IOI (A), the magnetizations of both GO–IO hybrids reach saturation and their magnetic properties were found to be good. The superior magnetic property of GO–IOF in comparison to GO–IOI can also be correlated to the iron content in both GO–IO, as evident from the results of elemental analysis. The percentage of iron bound to GO was higher in the case of GO–IOF as compared to GO–IOI which may be correlated to the fact that the particle size of an iron nanoparticle present in ferrofluid was much smaller as compared to iron nanoparticles formed in GO–IOI. The smaller size of nanoparticles provides space for the inclusion of the higher percentage
Fig. 4. Surface morphology of GO (A), GO–IOI (B), GO–IOF (C) and ferrofluid (D).
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Fig. 5. a.Elemental analysis of GO. b. Elemental analysis of GO–IOF (A) and GO–IOI (B).
of iron as the void space present between iron nanoparticles bound on the GO is minimized. 3.4.1. In vitro cell viability assay Cell viability was assessed using MTT assay. The result is shown in Fig. 8. The results of the present study demonstrated that dispersion of ANS loaded GO–IO resulted in a decrease in cell viability as compared to ANS. Pure ANS and GO showed 16.6% and 13.1% inhibition of cell viability respectively. Between GO–IO and GO–IOF, GO–IOF showed higher inhibition of cell viability as compared to GO–IOI which may be correlated to the small size of GO–IOF in comparison to GO–IOI. Another aspect of GO–IOF which can be correlated to its higher anti-proliferative activity is the number of IO nanoparticles conjugated on the GO. Due to the smaller size of IO nanoparticles present in a ferrofluid, higher number of IO nanoparticles could have been conjugated to GO. Apart from enhanced penetration due to smaller size, another aspect of IO which contributes to the enhanced anti-proliferative activity of GO–IO and GO–IOF as compared to ANS is the inherent anti-proliferative activity
of IO itself. According to recently published literature, it has been demonstrated that the magnetic IO nanoparticles show good antiproliferative activity against breast cancer MCF-7 cells [28]. Although the present results showed good antiproliferative activity of magnetic nanocomposites as compared to pure drug, the concentration of magnetic nanocomposites required can still be decreased. The particle size can be further decreased along with an increase in net positive charge for enhancing the anti-proliferative activity of the fabricated magnetic nanocomposites using the minimum quantity of drug loaded magnetic nanocomposites which will be much better from a patient's point of view. The loading of drug can also be increased for decreasing net concentration of magnetic nanocomposites. The overall result suggests that the inhibition of cell viability was dose dependent. The present findings give a clear outline about the superiority of GO magnetic nanocomposites for treatment of breast cancer as compared to plain ANS. The local hyperthermia caused by IO can play a vital role in enhancing its antiproliferative activity which in turns depends on the magnetic susceptibility, size and density of IO near tumor cells. From the study, we can
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Fig. 6. Magnetic activity of GO–IOF (A—side view, C—aerial view) and GO–IOI (B—side view, D—aerial view).
Fig. 7. Magnetic hysteresis loop of GO–IOI (A) and GO–IOF (B).
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Fig. 8. Cell viability assay of GO, ANS, GO–IOI, GO–IOF, GO–IOFA, GO–IOIA and ADR.
conclude that the loading of ANS on GO–IO enhances the anticancer activity, which means that with increasing solubility and stability in physiological solutions, the GO nanocarriers could indirectly enhance the dissolution of water insoluble anticancer drugs and so is the case with anticancer activity.
4. Conclusions In the present work, we have reported the synthesis of graphene based GO–IO, using two different methods and ferrofluid method was found to be superior with respect to iron content along with magnetic property of prepared GO–IO. Both, the GO–IOIA (74.3%) and GO–IOFA (84%) showed high loading efficiency. Particle size of GO–IOF was lesser than GO–IOI which may have effects on cellular uptake and ultimately anticancer activity. This fact has been confirmed by the results of cell viability assay in which GO–IOFA exhibited much higher cytotoxicity to MCF-7 cells than GO–IOIA and pure ANS. Taken together, this study demonstrates the viability of utilizing GO–IO as nanocarriers for targeted delivery of drugs, which may have potential clinical advantages pertaining to increased therapeutic efficacy and decreased local toxicity.
Acknowledgments The authors gratefully acknowledge Asbury Carbons, New Jersey for generously gifting graphite. We gratefully acknowledge Ferro-Tech Pvt. Ltd., USA for providing the gift samples of ferrofluid. We are also thankful to ACTREC, Mumbai (India) and SAIF, Indian Institute of Technology, Madras for carrying out in-vitro cell viability assay and VSM analysis, respectively.
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