Cancer is the uncontrolled growth of tissues and their rapid invasion without normal development and differentiation of cells [1
]. Although it is necessary to minimize side effects and improve efficiency to devise a successful cancer treatment regime, it is also important to address the major limitations of several therapeutic agents such as poor solubility, rapid deactivation, unfavorable pharmacokinetics and limited biodistribution [1
]. Nanomaterials have been widely studied for biomedical applications due to their high surface area-to-volume ratio, specific targeting capabilities and enhanced interaction with cells [1
]. Nanomaterials have enhanced properties such as absorption, scattering, magnetic resonance or even their effective capability of delivering drugs, as compared to the bulk form of materials. Hence, various nanomaterials have also been tested for cancer treatment [1
In contrast to chemotherapy and radiation, magnetic nanoparticles offer facile and effective cancer therapy treatment via hyperthermia with minimal side effects. Hyperthermia exposes malignant cells to heat and high temperatures (45–47 °C) as a means of weakening or destroying cancer cells [5
]. As temperatures in this range cause functional and structural impairment to cancer cells, such as protein damage, it eventually triggers apoptosis in these cells [6
]. Conventional hyperthermia employs an “outside-in” method of heating, where the majority of the heat is focused on the body’s surface, which then decreases in intensity as it moves away from the source [5
]. The energy from the external radiation in conventional hyperthermia is dispersed and does not discriminate thermally between healthy or cancer tissues, and thus, it causes hot spot regions that may trigger a relapse of cancer [5
]. Thermal discrimination is achieved when hyperthermia reaches only target malignant cells without causing any harm to healthy tissue [5
]. The major advantage of (nano) magnetic hyperthermia is the thermal differentiation between healthy and cancer tissues. This thermal differentiation in tissues is attributed to the targeting capability of magnetic nanoparticles that could enter small areas and be guided via external magnetic fields [5
]. Nanoparticles are used for targeted hyperthermia as they are able to reverse the direction of heat loss from outside-in to inside-out, making them the primary heating source in the modern hyperthermia method [5
]. The two main heat generation mechanisms by magnetic nanoparticles are relaxation and hysteresis losses, which are crucial to understanding their effect on magnetic hyperthermia therapy. Magnetic hyperthermia therapy relies on the physical phenomenon of magnetic losses, which takes place when an alternating magnetic field (AMF) is applied to the sample. Magnetic and structural factors such as the particle size (d), saturation magnetization (Ms
), the viscosity of the sample (η) as well as the anisotropy constant (Keff
) are the major parameters that influence magnetic losses [8
Research on nanomaterials, specifically superparamagnetic iron oxide nanoparticles (SPIONs) such as Fe3
], has been extensively carried out for targeted drug delivery, medical imaging, cell targeting and hyperthermia therapy [3
]. It was also clinically approved for human use [1
]. SPIONs were chosen for their excellent biocompatibility and magnetic properties [1
], with Fe3
nanoparticles favored for their high saturation magnetization [11
], as the saturation magnetization was also proportional to the heating efficiency for magnetic hyperthermia [12
]. The size of SPIONs is a crucial factor influencing its magnetic properties, with nanoparticles around 15–30 nm (average of 20 nm) required for superparamagnetism [12
]. A significant challenge facing iron oxide magnetic nanoparticles is that the smaller the nanoparticles, the lower the saturation magnetization exhibited. SPIONs with high saturation magnetization capacities are desired in order to apply lower AMF during magnetic hyperthermia [12
]. The morphological control of SPIONs, particularly nanocubes, allowed higher magnetization at small sizes than spherical shapes, even at low concentrations [12
]. Other challenges for using SPIONs that need to be addressed include concerns over toxicological properties, long-term impact on human health [2
] and the stability of the nanoparticle colloids due to their surface charge [9
]. Although there are issues regarding the safety of SPIONs for clinical use, it is an interchange between toxicity and overall health benefits, and many factors contribute to this fact, as this study shows. The distribution of SPIONs is determined by charge, which also affects its internalization in the target cells [9
]. In order to address these toxicity concerns and overcome the issue of aggregation, surface coating on the nanoparticles is employed.
Multifunctional properties were achieved using core-shell nanoparticles, where the shell (coating) could be used as a protective shield for the core or functionalized in a way that would reduce the cytotoxicity of the core [10
]. Coating nanoparticles with biocompatible and biodegradable materials ensures the elimination of undesirable immune reactions that might be triggered by the toxicity of a material. Moreover, the best solution to bring the magnetic core-shell nanoparticles to a more isotropic state in a zero-magnetic field is to provide a coating around the particles [10
]. SPIONs can be functionalized with many biocompatible polymers such as poly-(ethylene glycol) (PEG), dextran and polysaccharides to achieve better biocompatibility and stability in the blood [1
]. Other nanomaterials such as graphene and its derivatives, including reduced graphene oxide (RGO), have been used with SPIONs as nanocomposites with enhanced hyperthermia efficiency [14
]. The graphene family showed great potential in areas, such as gene delivery, loading anticancer drugs and antibacterial purposes, due to their enhanced surface properties and hydrophobic interactions [15
]. The use of biocompatible 2D nanomaterials such as graphene to develop SPION nanocomposites is another strategy to overcome the challenges facing SPIONs and enhance their effectiveness. Multifunctional iron oxide nanomaterials using RGO have also been synthesized for application in photothermal therapy due to their high NIR absorbance [16
]. The synergic effects of Fe3
/RGO were demonstrated by the heating profiles of SPIONs/RGO nanocomposites for in vitro magnetic hyperthermia that showed 90% killing efficiency when exposed to an AMF. These nanocomposites also had enhanced drug release properties in acidic environments, as RGO was sensitive to pH due to residual carboxylic functional groups, which allowed for a pH-responsive release of chemotherapeutic drugs to the cancer cells. Thus, the nanocomposite had enhanced drug delivery properties, and hence, were potential candidates for therapeutic carriers and hyperthermia agents [17
]. However, concerns over the toxicity of graphene and its derivatives have challenged the practical implementation of these nanocomposites. The graphene family, including graphene oxide (GO), is well known for unique interfaces with various functionalization sites that have enabled efficient drug delivery and diagnostics. Although the supposed cytotoxic effects of this family may lower its biocompatibility, it was found that the toxicity of GO depends on the concentration, the number of layers, density of oxygenation and cell-specific interaction [18
]. Cell viability assays determined that pristine GO showed higher cytotoxicity effects than GO functionalized with Fe3
]. On the other hand, another factor that restrained toxicity was lower GO concentration, as studies seemed to agree that lower amounts of GO (<4 μg/mL) showed no toxicity [19
]. Based on the literature, this work attempted to develop stable nanocube-like Fe3
NPs with graphene nanocomposites to capitalize on the synergistic effects between graphene and SPIONs for enhanced hyperthermia efficiency and possible drug delivery. The synthesized nanocomposites were then coated with PEG for enhanced biocompatibility. Moreover, the RGO sheets encapsulated the SPIONs as a means of protection from the acidic pH, while retaining their magnetization. The Fe3
nanocomposites were synthesized via a facile microwave hydrothermal synthesis for magnetic hyperthermia, with their sizes controlled within the required range for SPIONs as per literature (15–30 nm), by varying multiple synthesis parameters. The biocompatibility of the nanocomposites evaluated through cytotoxicity studies on human kidney cells and breast cancer cells showed cell viability of >70%, thus indicating its applicability for in vivo studies. The performance of these nanocomposites for application in magnetic hyperthermia was investigated in various dispersion media with promising SAR values of 58.33 W/g obtained in acidic pH solutions.
2. Experimental Methods
The chemicals used in the synthesis of Fe3O4 were Iron(III) Chloride (FeCl3), reagent grade 97% (Sigma-Aldrich, St. Louis, MO, USA); Poly-(ethylene glycol) (PEG), average molecular weight 200 (Sigma-Aldrich, St. Louis, MO, USA); Hydrazine Hydrate (N2H4), 80% (Loba Chemie, Mumbai, India); and MilliQ H2O. RGO synthesis included the use of Graphite fine powder, extra pure (Merck, Darmstadt, Germany); Sulfuric acid (H2SO4), 95% (Sigma-Aldrich, St. Louis, MO, USA); Sodium Nitrate PRS (NaNO3) (Sigma-Aldrich, St. Louis, MO, USA); Potassium Permanganate (KMnO4), extra pure (Sigma-Aldrich, St. Louis, MO, USA); Hydrogen Peroxide (H2O2), 30% (Sigma-Aldrich, St. Louis, MO, USA); and Hydrochloric acid (HCl), 37% (Sigma-Aldrich, St. Louis, MO, USA). On the other hand, the dispersion medium used for hyperthermia tests includes Phosphate Buffered Saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA); Buffer solution (acetic acid/sodium acetate), pH (20 °C) = 4.66 ± 0.01 (Merck, Darmstadt, Germany); and Dimethyl Sulphoxide (DMSO) (C2H6OS) (Loba Chemie, Mumbai, India). Chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA), Merck (Darmstadt, Germany) and Loba Chemie (Mumbai, India) and were used without any purification.
The Fe3O4 graphene nanocomposites were prepared by microwave hydrothermal synthesis in CEM Mars 6 microwave (CEM, Charlotte, NC, USA) under varying conditions of concentration, pressure, temperature, power and time, as given below.
2.1. Fe3O4 Nanoparticles (SPIONs) Synthesis
In a 25 mL beaker, 70 mM (90.8 mg) FeCl3 was mixed with 8 mL MilliQ water and magnetically stirred for 10 min at room temperature. Then, stirring was switched off, and 1 mL hydrazine hydrate was added. The samples were quickly transferred to CEM vessels and subjected to microwave irradiation under specific synthesis parameters: 900 W, 250 psi, 200 °C and 10 min. After the reaction, the samples were washed with deionized (DI) water in the centrifuge for five rounds under 3400 rpm, with 15 min per wash. The samples were then dried in the oven overnight at 80 °C.
2.2. Graphene Oxide (GO) Synthesis
The modified Hummer’s method was used to obtain graphene oxide (GO) [17
]. Briefly, 125 mL of concentrated sulfuric acid was mixed with 2 g graphite at 0 °C. Then, 2.5 g of sodium nitrate was slowly mixed in, followed by the addition of 20 g of potassium permanganate while maintaining the temperature below 20 °C. The mixture was then heated to 35 °C for 2 h with vigorous stirring, followed by the addition of 230 mL of DI water with the temperature kept below 50 °C. The reaction was terminated with 20 mL of 30% hydrogen peroxide with subsequent color change to yellow. The product obtained was then washed with 100 mL of 10% hydrogen chloride solution, followed by extensive centrifuge washing with hot MilliQ water. The washing process continued until the pH of GO was near neutral. Sonication of the sample was performed for 10 min at a power of 6 in bath sonication (VWR®
Ultrasonic Cleaner, Avantor, Radnor, PA, USA). The sample was then dried at 60 °C overnight.
2.3. Fe3O4/RGO Nanocomposites Synthesis
The reduced graphene oxide (0.1% RGO) for the nanocomposites was derived from the addition of a homogenous aqueous GO solution (2 mg/mL) that was prepared by bath sonication to the mixture obtained in Fe3O4 synthesis. The initial procedure for nanocomposite synthesis was similar to that of Fe3O4 nanoparticles. After obtaining 70 mM solution of iron chloride, specific amounts of GO stock solution were added to obtain the desired weight percentage of Fe3O4 in the nanocomposite. The samples were sonicated for 60 min for complete exfoliation of GO and its interaction with the iron hydroxide flocs. Then, 1 mL hydrazine hydrate was added, and the samples were quickly transferred to CEM vessels and placed inside the CEM MW under optimized synthesis parameters: 900 W, 250 psi, 200 °C and 10 min. Samples were washed in the centrifuge as stated earlier, followed by oven drying at 80 °C overnight. For nanocomposites with PEG, an optimized amount of 8 mL PEG 200 was added after the GO solution during synthesis.
The nanomaterials synthesized were characterized using X-ray diffraction (XRD) (Rigaku Miniflex 600, The Woodlands, TX, USA) and high-resolution transmission electron microscopy (HR-TEM JEM-2100F, JEOL, Boston, MA, USA). A copper grid was used for the HR-TEM sample preparation. The average particle sizes were estimated from the peaks obtained in the X-ray diffraction using the Scherrer equation.
2.5. Procedures for Application Testing
Cytotoxicity testing in vitro for biocompatibility confirmation was conducted on two cell lines: Breast cancer cell line, MCF-7 (Michigan Cancer Foundation–7), and Embryonic kidney cell line, HEK-2 (Human Embryonic Kidney–293). The cells were purchased from (ATCC, Manassas, VA, USA). Cell culturing started with the preparation and aspiration of 1% penicillin/streptomycin (P/S) and 10% fetal bovine serum (FBS) media followed by its filtration for cell passaging with the addition of trypsin. The cell passaging process was initiated by aspirating the media from its original flask to a clean flask that was washed with PBS. It included the addition of trypsin, incubation, the addition of media, centrifugation, aspirating the supernatant and mixing the culture media along with the remaining pellet until homogeneous. After that, cell counting was conducted, where equal amounts of the cell suspension and Trypan blue were mixed together in order to dye the dead cells. The nanocomposites to be tested for cytotoxicity were suspended in media (10% FBS, 90% DMEM and 1% Penicillin) at a concentration of 200 µg/mL and was then added to the culture media prior to incubation for 24–48 h.
2.5.2. Magnetic Hyperthermia
Easy Heat from Ambrell was used to test for magnetic hyperthermia. The SPIONs solution was placed in a glass tube at the center of a magnetic induction coil (8 turns, 3.7 cm diameter), held by a stand with a metal clamp having a plastic insulation cover to avoid any noise (heating effects) from the clamp itself. The trials were carried out by the application of the magnetic field in pulses with the field switched on for 5 min followed by the field switched off period for 10 s. The overall time of magnetic induction did not exceed 30 min per complete session. The obtained heating curves were fitted with a second degree polynomial model to estimate the initial slope (ΔT/Δt) used in the calculation of SAR value. For the SPIONs to be tested for hyperthermia therapy, a suitable medium must be used to provide an accurate simulation of the environment of cancer cells, preferably with minimal effects of particle aggregation. Thus, PBS (phosphate buffered saline), aqueous DMSO (dimethyl sulfoxide) solution (1:1) (DMSO:water) [20
] and a pH buffer (pH = 4.66) were chosen as the media for the hyperthermia tests. Samples tested include Fe3
/RGO and PEG-coated Fe3
/RGO labelled Fe3
/RGO/PEG. The dispersion media without any nanoparticles were tested as controls in this test.