Carbon-derived nanoparticles (NPs) such as single- and multi-walled carbon nanotubes, fullerenes, and graphene are all receiving attention because of their interesting and unusual electronic
], and mechanical
] properties. We have recently demonstrated a facile route towards the synthesis of nanosized water-soluble sulfonated graphene sheets (SGSs) that use graphite as the starting material
]. This method relies on the addition of phenyl radicals with subsequent sulfonation of the phenyl groups and produces fewer defects and holes that can be introduced into the graphene plates through the use of heavy sonication. A possible application of these SGSs is within the medical sector due to their enhanced solubility (compared to other graphene derivatives) and potential for surface modifications for attachment of biomolecules and drugs. However, the interaction of SGSs with biological systems has yet to be investigated and is the basis of the work described herein.
To date, much of the biological work regarding graphene has focused on assessing the cytotoxicity, cell adhesion, proliferation, and antibacterial properties of graphene oxide
] as well as biodistribution, toxicology, and internalization of various suspensions of GO complexes. These include 125
I and 188
Re radioisotope-labeled GO
], PEGylated GO for cellular imaging and delivery of water-insoluble cancer drugs
], and the imaging and treatment of brain, lung, and breast xenograft tumors in mice through the use of photothermal light therapy from the absorption of near-infrared (NIR) light by PEGylated GO with fluorescent Cy7 probes
Toxicity analysis (in vitro
) of GO (prepared using chemical vapor deposition or the modified Hummers method
]) on lung
] and neuronal
] cell lines (A549 and PC12, respectively) has shown concentration-dependent cytotoxicity. The exact mechanism of cell death from GO remains uncertain although a slight increase in lactate dehydrogenase (LDH) from cells, generation of reactive oxygen species, and weak activation of a caspase-3-mediated apoptosis pathway have all been reported. These reports suggest GO cytotoxicity from either direct cellular membrane damage or activation of natural cellular suicide mechanisms.
Similarly, in vivo
mouse toxicology studies have shown that GO nanoplatelets of diameters 10 to 700 nm apparently cause no acute toxicities at low doses
]. However, at high doses (10 mg/kg), significant pathological changes such as granulomatous lesions, pulmonary edema, inflammatory cell infiltration, and fibrosis were observed throughout the lungs.
In light of the potential applications of graphene materials in drug delivery, imaging, and thermal therapy, but with limitations due to cytotoxicity of GO, we sought to investigate the in vitro interaction of our highly water-soluble SGS with liver cancer cells. Our initial studies using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), WST-1[2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1), and LDH colorimetric assays have shown that SGSs are non-toxic up to concentrations of 10 μg/ml. We also show that liver cancer cell lines (SNU449 and Hep3B) can internalize SGSs of diameters up to 5 μm, which in some cases are comparable to the size of the cells themselves. Preliminary electron microscopy analysis also suggests that these cells are capable of folding and compartmentalizing sheets of smaller sizes (approximately 1.41 μm) although more work should be undertaken to validate.
Since graphene has been documented to be the hardest material known
], this unique behavior of water-soluble SGS with cells is counterintuitive and suggests a novel finding that may have far-reaching applications in biology and medicine such as enhanced drug delivery (due to the large graphene surface area), and should warrant further investigation. Given that these SGSs are non-toxic up to 10 μg/ml, we feel they can be used as an adequate scaffold to simultaneously attach targeting moieties such as EGFR antibodies (e.g., cetuximab, C225) and chemo-agents such as doxorubicin and gemcitabine in a bid to treat hepatocellular carcinoma legions. The use of a targeted thermal ‘trigger’ such as photon activation (i.e., NIR light) or radiofrequency electric fields could allow these SGSs to release their cargo into the cells upon irradiation by a stimuli. Such a scheme has recently been reported using cisplatin-filled ultra-short carbon nanotubes that release their cargo upon exposure to high-intensity radiofrequency electric fields